NOVEL CELL THERAPY SYSTEM

NOUVEAU SYSTEME DE THERAPIE CELLULAIRE

English Abstract

The present invention relates to therapeutics, compositions, kits and methods of treatment comprising: (a) an immune cell or progenitor thereof, expressing a receptor comprising an antigen-recognition domain and a signalling domain, wherein the antigen-recognition domain binds to a tumour-specific antigen, preferably a dysfunctional P2X7 receptor, expressed on a cell surface; and (b) a bridging molecule comprising: (i) a targeting moiety that binds to a cell surface molecule on a target cell; and (ii) a tumour-specific epitope moiety, preferably a dysfunctional P2X7 receptor epitope moiety, that is bound by the antigen recognition domain.

French Abstract

La présente invention concerne des agents thérapeutiques, des compositions, des kits et des méthodes de traitement comprenant : (a) une cellule immunitaire ou un progéniteur de celle-ci, exprimant un récepteur comprenant un domaine de reconnaissance d'antigène et un domaine de signalisation, le domaine de reconnaissance d'antigène se liant à un antigène spécifique d'une tumeur, de préférence un récepteur P2X7 dysfonctionnel, exprimé sur une surface cellulaire; et (b) une molécule de pontage comprenant : (i) une fraction de ciblage qui se lie à une molécule de surface cellulaire sur une cellule cible; et (ii) une fraction d'épitope spécifique de tumeur, de préférence une fraction d'épitope de récepteur P2X7 dysfonctionnel, qui est liée par le domaine de reconnaissance d'antigène.

Claims

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CLAIMS
1. A two component therapeutic comprising:
(a) an immune cell or progenitor thereof, expressing a receptor comprising
an
antigen-recognition domain and a signalling domain, wherein the antigen-
recognition
domain binds to a tumour-specific antigen expressed on a cell surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on a target cell;

and
(ii) a tumour-specific antigen epitope moiety that is bound by the antigen
recognition domain.
2_ A two component therapeutic of claim 1, wherein the tumour
specific antigen an
antigen expressed on a solid tumour.
3. A two component therapeutic of claim 1, wherein the tumour
specific antigen is
any one of nfP2X7, EGFRylll or CLDN6.
4. A two component therapeutic comprising:
(a) an immune cell or progenitor thereof, expressing a receptor comprising
an
antigen-recognition domain and a signalling domain, wherein the antigen-
recognition
domain binds to a dysfunctional P2X7 receptor expressed on a cell surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on a target cell;

and
(ii) a dysfunctional P2X7 receptor epitope moiety that is bound by the antigen

recognition domain.
5. A composition comprising:
(a) an immune cell or progenitor thereof, expressing a
receptor comprising an
antigen-recognition domain and a signalling domain, wherein the antigen-
recognition
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domain binds to a tumour-specific antigen, preferably a dysfunctional P2X7
receptor,
expressed on a cell surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on a target cell;

and
(ii) a tumour-specific antigen epitope moiety, preferably a dysfunctional P2X7
receptor epitope moiety, that is bound by the antigen recognition domain.
6. A kit comprising:
(a) an immune cell or progenitor thereof, expressing a receptor comprising
an
antigen-recognition domain and a signalling domain, wherein the antigen-
recognition
domain binds to a tumour-specific antigen, preferably a dysfunctional P2X7
receptor,
expressed on a cell surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on a target cell;

and
(ii) a tumour-specific antigen epitope moiety, preferably a dysfunctional P2X7
receptor epitope moiety, that is bound by the antigen recognition domain.
7. The therapeutic, composition or kit according to any one
of claims 1 to 6,
wherein the bridging molecule is a polypeptide.
8. The therapeutic, composition or kit according to claim 7,
wherein the
polypeptide is expressed by the immune cell or progenitor thereof.
9. The therapeutic, composition or kit according to any one
of claims 1 to 6,
wherein the bridging molecule is a polypeptide, and the polypeptide is
provided in the
therapeutic, composition or kit, or is encoded by a nucleic acid provided in
the
therapeutic, composition or kit.
10. An immune cell, or progenitor thereof comprising:
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(i) a chimeric antigen receptor comprising an antigen-recognition domain
that binds a first tumour antigen, preferably wherein the first tumour
antigen is a dysfunctional P2X7 receptor, and
(ii) an inducible expression construct encoding a bridging molecule in the
form of a fusion protein comprising (a) an antibody, or antigen binding
fragment thereof, that binds a second tumour antigen, and (b) a peptide
or a fragment of the first tumour antigen, preferably the dysfunctional
P2X7 receptor.
11. A bridging molecule comprising:
a targeting moiety that binds to a cell surface molecule on a target cell;
and
(ii) a tumour-specific antigen epitope moiety, preferably a
dysfunctional P2X7
receptor epitope moiety, that is bound by an antigen recognition domain.
12. The bridging molecule of claim 11, wherein the molecule is in the form
of a
fusion protein.
13. A nucleic acid comprising a nucleotide sequence encoding a bridging
molecule as described in any one of claims 1 to 12.
14. The nucleic acid of claim 13, wherein the nucleic acid comprises a
first
nucleotide sequence encoding the targeting moiety and a second nucleotide
sequence
encoding the tumour-specific antigen epitope moiety, preferably a
dysfunctional P2X7
receptor epitope moiety.
15. A vector or expression construct comprising a nucleic acid of claim 14.
16. The vector or expression construct of claim 15, wherein the vector or
construct
further comprises a nucleic acid sequence encoding the chimeric antigen
receptor
comprising an antigen-recognition domain that binds to a dysfunctional P2X7
receptor.
17. A method of treating a disorder in a subject, the method comprising
administering to the subject:
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(a) a cell expressing a receptor comprising an antigen-recognition domain
and a
signalling domain, wherein the antigen-recognition domain binds to a tumour-
specific
antigen, preferably a dysfunctional P2X7 receptor, expressed on a cell
surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface rnolecule on a target
cell; and
(ii) a tumour-specific antigen epitope moiety, preferably a dysfunctional P2X7
receptor epitope moiety, that is bound by the antigen recognition domain,
thereby treating the disorder in the subject.
18. A method of treating cancer in a subject, the method comprising
administering:
(a) an immune cell expressing a receptor cornprising an antigen-recognition

domain and a signalling domain, wherein the antigen-recognition domain binds
to a
tumour-specific antigen, preferably a dysfunctional P2X7 receptor, expressed
on a cell
surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface rnolecule on a target
cell; and
(ii) a tumour-specific antigen epitope moiety, preferably a dysfunctional P2X7

receptor epitope moiety, that is bound by the antigen recognition domain.
19. A method of killing a target cell, the method including exposing the
target cell
to:
(a) an immune cell expressing a receptor comprising an antigen-recognition
domain and a signalling domain, wherein the antigen-recognition domain binds
to a
tumour-specific antigen, preferably a dysfunctional P2X7 receptor, expressed
on a cell
surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on the target
cell; and
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(ii) a tumour-specific antigen epitope moiety, preferably a dysfunctional P2X7

receptor epitope moiety, that is bound by the antigen recognition domain,
thereby killing the target cell.
20. The method of claim 19, wherein the target cell is a cancer cell, or a
cell on
which an MHC I or II molecule presents a peptide from an infectious agent.
21. The rnethod of clairn 19 or 20, wherein the target cell does not
express
dysfunctional P2X7 receptor.
22. The method of any one of claims 17 to 21, wherein 2 or more bridging
molecules are administered to a subject, each bridging molecule comprising a
targeting
moiety that binds to a different cell surface molecule on a target cell.
23. The method of any one of claims 17 to 22, wherein the bridging molecule
is
delivered via infusion to the subject.
24. The method of any one of claims 17 to 22, wherein the bridging molecule
is a
polypeptide encoded by a nucleic acid sequence that is expressed by the immune
cell
expressing the chimeric antigen receptor.
25. The method of claim 24, wherein the nucleic acid sequence comprises an
inducible promoter for enabling inducible expression thereof.
26. The therapeutic, composition or kit of any one of claims 1 to 9, the
immune cell
of claim 10, the bridging molecule of claims 11 or 12, or the method of any
one of claims
17 to 25, wherein the bridging molecule comprises targeting moieties for more
than one
class of cell surface molecule on a target cell.
27. The therapeutic, composition or kit of any one of claims 1 to 9, the
immune cell
of claim 10, the bridging molecule of claims 11 or 12, or the method of any
one of claims
17 to 26, wherein the bridging molecule comprises targeting moieties for more
than one
epitope of the same cell surface molecule on a target cell.
28. The therapeutic, composition or kit of any one of claims 1 to 9, the
immune cell
of claim 10, or the method of any one of claims 17 to 27, wherein the antigen-
recognition domain of the CAR binds to an epitope associated with an adenosine

triphosphate (ATP)-binding site of the dysfunctional P2X7 receptor.
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29. The therapeutic, composition or kit, the immune cell, or the method of
claim
28, wherein the dysfunctional P2X7 receptor has a reduced capacity to bind ATP
at the
ATP-binding site compared to an ATP-binding capacity of a functional P2X7
receptor
(e.g., a receptor having wild-type sequence and having a conformation or fold
of an
ATP-binding receptor).
30. The therapeutic, composition or kit, the immune cell, or the method of
claim
29, wherein the dysfunctional P2X7 receptor cannot bind ATP at the ATP-binding
site.
31. The therapeutic, composition or kit, the immune cell, or the method of
any one
of claims 28 to 30, wherein the dysfunctional P2X7 receptor has a
conformational
change that renders the receptor dysfunctional.
32. The therapeutic, composition or kit, the immune cell, or the method of
claim
31, wherein the conformational change is a change of an amino acid from the
trans-
conformation to the cis-conformation.
33. The therapeutic, composition or kit, the immune cell, or the method of
claim
32, wherein the amino acid that has changed from a trans-conformation to a cis-

conformation is proline at amino acid position 210 of the dysfunctional P2X7
receptor.
34. The therapeutic, composition or kit, the immune cell, or the method of
any one
of any one of claims 28 to 33, wherein the antigen-recognition domain binds to
an
epitope that includes the proline at amino acid position 210 of the
dysfunctional P2X7
receptor.
35. The therapeutic, composition or kit, the immune cell, or the method of
claim
34, wherein the antigen-recognition domain binds to an epitope that includes
one or
more amino acid residues spanning from glycine at amino acid position 200 to
cysteine
at amino acid position 216, inclusive, of the dysfunctional P2X7 receptor.
36. The therapeutic, composition or kit, the immune cell, or the method of
any one
of claims 28 to 30, wherein the antigen-recognition domain of the CAR
comprises amino
acid sequence homology to the amino acid sequence of an antibody, or a
fragment
thereof, that binds to the dysfunctional P2X7 receptor.
37. The therapeutic, composition or kit, the immune cell, or the method of
claim
36, wherein the antigen-recognition domain has amino acid sequence homology to
the
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amino acid sequence of a fragment-antigen binding (Fab) portion of an antibody
that
binds to a dysfunctional P2X7 receptor.
38. The therapeutic, composition or kit, the imrnune cell, or the method of
claim 35
or 37, wherein the antigen-recognition domain comprises a single-chain
variable
fragment (scFv), or a multivalent scFv or a single-antibody domain (sdAb) that
binds to
a dysfunctional P2X7 receptor.
39. The therapeutic, composition or kit, the immune cell, or the method of
any one
of claims 26 to 35 wherein the signalling domain includes a portion derived
from a co-
stimulatory receptor, optionally wherein the signalling domain includes a
portion derived
from an activation receptor and a portion derived from a co-stimulatory
receptor.
40. The therapeutic, composition or kit, the immune cell, or the method of
claim
36, wherein the co-stimulatory receptor is selected from the group consisting
of CD27,
CD28, CD30, CD40, DAP10, OX40, 4-1BB (CD137) and ICOS.
41. The therapeutic, composition or kit, the immune cell, or the method of
any one
of claims 28 to 35 wherein the receptor comprising the antigen-recognition
domain and
signalling domain is a chimeric antigen receptor (CAR).
42. The therapeutic, composition or kit of any one of claims 1 to 9, the
immune cell
of claim 10, the bridging molecule of claims 11 or 12, or the method of any
one of claims
17 to 25, wherein the targeting moiety of the bridging molecule that binds to
a cell
surface molecule on a target cell comprises or consists of a peptide or
antibody or
antibody fragment.
43. The therapeutic, composition or kit of any one of claims 1 to 9, the
immune cell
of claim 10, the bridging molecule of claims 11 or 12, or the method of any
one of claims
17 to 25, wherein the targeting moiety of the bridging molecule comprises a
ligand or
binding partner for a protein or receptor present on the target cell surface.
44. The therapeutic, composition or kit of any one of claims 1 to 9, the
immune cell
of claim 10, the bridging molecule of claims 11 or 12, or the method of any
one of claims
17 to 25, wherein the cell surface molecule bound by the targeting molecule is
an
antigen, preferably an antigen as described herein.
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45. The therapeutic, composition or kit of any one of claims 1 to 9, the
immune cell
of claim 10, the bridging molecule of claims 11 or 12, or the method of any
one of claims
17 to 25, wherein the cell surface molecule bound by the targeting molecule is
selected
from a protein, a lipid moiety, a glycoprotein, a glycolipid, a carbohydrate,
a
polysaccharide, a nucleic acid, an MHC-bound peptide, or a combination
thereof.
46. The therapeutic, composition or kit of any one of claims 1 to 9, the
immune cell
of claim 10, the bridging molecule of claims 11 or 12, or the method of any
one of claims
17 to 25, wherein the targeting moiety of the bridging molecule comprises a
targeting
antibody or antibody fragment.
47. The therapeutic, composition or kit, the immune cell, the bridging
molecule, or
the method of claim 46, wherein the targeting antibody or antibody fragment is
an
immunoglobulin (Ig), optionally selected from an lgG, an IgA, an IgD, an IgE,
an lgM, a
fragment thereof or a modification thereof.
48. The therapeutic, composition or kit, the immune cell, the bridging
molecule, or
the method of claim 46 or 47, wherein the targeting antibody or fragment
thereof is an
lgG1.
49. The therapeutic, composition or kit, the immune cell, the bridging
molecule, or
the method of any one of claims 46 to 48, wherein the targeting antibody or
fragment
thereof binds to an antigen on a cancer cell.
50. The therapeutic, composition or kit, the immune cell, the bridging
molecule, or
the method of claim 49, wherein the antigen on the cancer cell is a tumour
associated
antigen.
51. The therapeutic, composition or kit, the immune cell, the bridging
molecule, or
the method of claim 47, wherein the tumour associated antigen is selected from
the
group consisting of: 0D33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-3), CD44
(HCAM),
CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1), LeY, FRp, MICA/B, CD305 (LAIR-
1), CD366 (TIM-3), C096 (TACTILE), C0133, CD56, CD29 (ITGB1), CD44 (HCAM),
CD47 (IAP), CD66 (CEA), CD112 (Nectin2), CD117 (c-Kit), CD133, CD146 (MCAM),
CD155 (PVR), CD171 (LI CAM), CD200 (OX-2), CD221 (IGF1), CD227 (MUC1), CD243
(MRD1), CO246 (ALK), CD271 (LNGFR), CD19, CD20, GD2, and EGFR.
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52. The therapeutic, composition or kit of any one of claims 1 to 9, the
immune cell
of claim 10, the bridging molecule of claims 11 or 12, or the method of any
one of claims
17 to 25, wherein the dysfunctional P2X7 receptor epitope moiety of the
bridging
molecule is provided in the form of a P2X7 receptor, or a fragment of a P2X7
receptor
that has at least one of the three ATP binding sites that are formed at the
interface
between adjacent correctly packed monomers that are unable to bind ATP.
53. The therapeutic, composition or kit of any one of claims 1 to 9, the
immune cell
of claim 10, the bridging molecule of claims 11 or 12, or the method of any
one of claims
17 to 25, wherein the dysfunctional P2X7 receptor epitope moiety of the
bridging
molecule comprises or consists of a fragment of a dysfunctional P2X7 receptor.
54. The therapeutic, composition or kit, the immune cell, the bridging
molecule, or
the method of claim 53, wherein the fragment of a dysfunctional P2X7 receptor
is
selected from the amino acid sequences set forth in any one of SEQ ID NOs: 2
to 30
and 168.
55. The therapeutic, composition or kit, the immune cell, the bridging
molecule, or
the method of claims 53 or 54, wherein the dysfunctional P2X7 receptor epitope
moiety
is bound by an antibody that binds to dysfunctional P2X7 receptors, but is not
bound by
antibodies which bind to functional P2X7 receptors.
56. The therapeutic, composition or kit, the immune cell, the bridging
molecule, or
the method of any one of claims 52 to 55, wherein the bridging molecule
comprises 2 or
more dysfunctional P2X7 receptor epitope moieties.
57 The therapeutic, composition or kit, the immune cell, the
bridging molecule, or
the method of claim 56, wherein the 2 or more dysfunctional P2X7 receptor
epitope
moieties comprise or consist of the same sequence, or of different sequences.
58. The therapeutic, composition or kit, the immune cell, the bridging
molecule, or
the method of claim 57, wherein the bridging molecule comprises a
dysfunctional P2X7
receptor epitope moiety in the form of the E200 epitope and a further
dysfunctional
P2X7 receptor epitope moiety in the form of the E300 epitope.
59. The therapeutic, composition or kit, the immune cell, the bridging
molecule, or
the method of claim 57, wherein the bridging molecule comprises a
dysfunctional P2X7
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receptor epitope moiety in the form of the E200 epitope and a further
dysfunctional
P2X7 receptor epitope moiety in the form of the composite epitope.
60.
The therapeutic, composition or kit, the immune cell, the bridging
molecule, or
the method of claim 57, wherein the bridging molecule comprises a first
dysfunctional
P2X7 receptor epitope moiety in the form of the E200 epitope and a further
dysfunctional P2X7 receptor epitope moiety in the form of the E200 epitope.
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Description

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Novel Cell Therapy System
Field of the invention
[0001]
The present invention relates to chimeric antigen receptors, effector
cells
expressing an antigen receptor and methods of using antigen receptors for the
prevention and/or treatment of various conditions, including cancer.
Related applications
[0002] This application claims priority from Australian provisional
applications
AU 2021900708, AU 2021902565 and AU 2021902830, the contents of each of which
are hereby incorporated by reference in their entirety.
Background of the invention
[0003] Cancer immunotherapy is a rapidly growing field. The development of T
cells
expressing chimeric antigen receptors (CARs) has revolutionised adoptive cell
therapies.
[0004] The potential of this approach has been demonstrated in clinical
trials, wherein
CAR T cells were infused into adult and paediatric patients with B-cell
malignancies,
neuroblastoma, and sarcoma. To date, over 500 clinical trials have emerged
worldwide,
designed at testing the efficacy of CAR T cells targeted to bind 64 different
tumour
associated antigens. Among these, three CD19-specific CAR T cell products have
been
approved for the treatment of acute lymphoblastic leukaemia (ALL), large B
cell
lymphoma and mantle cell lymphoma. To date, most of the success with CAR T
therapies has been observed in the context of so-called "liquid" tumours, or
where the
CARs are directed to CD19, CD22 or the B cell maturation antigen (BCMA).
[0005] Several challenges remain in the clinical application of CAR T cell
therapies.
These include difficulties with achieving sufficient therapeutic responses in
the context
of solid tumours, which may be due to insufficient activation, expansion and
persistence
of CAR T cells and/or the immunosuppressive tumour microenvironment. In
addition,
the long-term clinical application of CAR T therapies can be negatively
impacted by the
selective pressure of mono-specific CAR T cells, antigen-negative escape
variants, and
antigen depletion. Moreover, low levels of target antigen expression in
healthy tissues
can result in severe "on-target, off-tumour" toxicities. Finally, cytokine
release syndrome
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(CRS) and CAR T cell-related encephalopathy syndrome (CRES) are frequently
observed side effects of CAR T cell therapy.
[0006] There is consequently a need for new and improved approaches to CAR,
preferably CAR-T, therapies.
[0007] Reference to any prior art in the specification is not an
acknowledgment or
suggestion that this prior art forms part of the common general knowledge in
any
jurisdiction or that this prior art could reasonably be expected to be
understood,
regarded as relevant, and/or combined with other pieces of prior art by a
skilled person
in the art.
Summary of the invention
[0008] In one aspect, the present invention provides a two component
therapeutic
cornprising:
(a) an immune cell or progenitor thereof, expressing a receptor comprising
an
antigen-recognition domain and a signalling domain, wherein the antigen-
recognition
domain binds to a tumour-specific antigen expressed on a cell surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on a target cell;

and
(ii) a tumour-specific antigen epitope moiety that is bound by the antigen
recognition domain.
[0009] In any aspect, the tumour-specific antigen is an antigen expressed on a
solid
tumour or liquid tumour. In one embodiment, the tumour-specific antigen is any
one of
nfP2X7, EGFRvIl I or CLDN6.
[0010] In one aspect, the present invention provides a two component
therapeutic
cornprising:
(a)
an immune cell or progenitor thereof, expressing a receptor comprising an
antigen-recognition domain and a signalling domain, wherein the antigen-
recognition
domain binds to a dysfunctional P2X7 receptor expressed on a cell surface; and
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(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on a target cell;

and
(ii) a dysfunctional P2X7 receptor epitope moiety that is bound by the antigen

recognition domain.
[0011] In another aspect, the present invention provides a composition
comprising:
(a) an immune cell or progenitor thereof, expressing a receptor comprising
an
antigen-recognition domain and a signalling domain, wherein the antigen-
recognition
domain binds to a dysfunctional P2X7 receptor expressed on a cell surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on a target cell;

and
(ii) a dysfunctional P2X7 receptor epitope moiety that is bound by the antigen

recognition domain.
[0012] In another aspect, the present invention provides a kit comprising:
(a) an immune cell or progenitor thereof, expressing a receptor comprising
an
antigen-recognition domain and a signalling domain, wherein the antigen-
recognition
domain binds to a dysfunctional P2X7 receptor expressed on a cell surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on a target cell;

and
(ii) a dysfunctional P2X7 receptor epitope moiety that is bound by the antigen

recognition domain.
[0013] In any embodiment, the bridging molecule may be a polypeptide, or a
polypeptide conjugated to a molecule with the function of a bridging molecule,
e.g. a
DNA aptamer. The polypeptide may be expressed by the immune cell or progenitor
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thereof. Alternatively, the therapeutic, composition or kit may comprise the
polypeptide,
or a nucleic acid encoding said polypeptide.
[0014] In a further aspect, the present invention provides an immune cell, or
progenitor thereof comprising:
(i) a receptor comprising an antigen-recognition domain and a signalling
domain, wherein the antigen-recognition domain binds a first tumour
antigen, wherein the first tumour antigen is a tumour-specific antigen
(preferably dysfunctional P2X7 receptor), and
(ii) an inducible expression construct encoding a bridging molecule in the
form of a fusion protein comprising (a) an antibody, or antigen binding
fragment thereof, that binds a second tumour antigen, and (b) a peptide
or a fragment of a tumour-specific antigen (preferably a dysfunctional
P2X7 receptor).
[0015] In another aspect, the present invention provides a bridging molecule
comprising:
(i) a targeting moiety that binds to a cell surface molecule on a target
cell; and
(ii) a tumour-specific antigen epitope moiety (preferably a dysfunctional
P2X7
receptor epitope moiety) that is recognised or capable of being bound by an
antigen
recognition domain of a receptor, optionally wherein the receptor is a
chimeric antigen
receptor.
[0016] The bridging molecule may be a polypeptide, for example a fusion or
chimeric
protein. In alternative embodiments, the bridging molecule may comprise
polypeptides
or peptides that are linked via linking molecules.
[0017] In another aspect, the present invention provides a nucleic acid
comprising a
nucleotide sequence encoding a bridging molecule as described herein.
Preferably, the
nucleic acid comprises a first nucleotide sequence encoding a targeting moiety
and a
second nucleotide sequence encoding a tumour-specific antigen epitope moiety.
Preferably, the tumour-specific antigen epitope moiety is a dysfunctional P2X7
receptor
epitope moiety.
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[0018] In another aspect, the present invention provides a vector or
expression
construct comprising a nucleic acid of the invention. In one embodiment, the
vector or
expression construct further comprises a nucleotide sequence encoding the
receptor
comprising an antigen-recognition domain and signalling domain, wherein the
antigen-
recognition domain recognises a dysfunctional P2X7 receptor expressed on a
cell
surface, as described herein.
[0019] In another aspect, the present invention provides a method of treating
a
disorder in a subject, the method comprising administering to the subject:
(a) a cell expressing a receptor comprising an antigen-recognition domain
and a
signalling domain, wherein the antigen-recognition domain binds to a tumour-
specific
antigen (preferably, the tumour specific antigen is a dysfunctional P2X7
receptor)
expressed on a cell surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on a target cell;
and
(ii) a tumour-specific antigen epitope moiety, preferably a dysfunctional P2X7
receptor epitope moiety, that is bound by the antigen recognition domain,
thereby treating the disorder in the subject.
[0020] In another aspect, the present invention provides a method of treating
cancer
in a subject, the method comprising administering:
(a) an immune cell expressing a receptor comprising an antigen-recognition
domain and a signalling domain, wherein the antigen-recognition domain binds
to a
tumour-specific antigen, preferably a dysfunctional P2X7 receptor, expressed
on a cell
surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on a target cell;
and
(ii) a tumour-specific epitope moiety, preferably a dysfunctional P2X7
receptor
epitope moiety, that is bound by the antigen recognition domain.
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[0021] In another aspect, the present invention provides a method of killing a
target
cell, the method including exposing the target cell to:
(a) an immune cell expressing a receptor comprising an antigen-recognition
domain and a signalling domain, wherein the antigen-recognition domain binds
to a
tumour-specific antigen, preferably a dysfunctional P2X7 receptor, expressed
on a cell
surface; and
(b) a bridging molecule comprising:
(i) a targeting moiety that binds to a cell surface molecule on the target
cell; and
(ii) a tumour-specific antigen epitope moiety, preferably a dysfunctional P2X7

receptor epitope moiety, that is bound by the antigen recognition domain,
thereby killing the target cell.
[0022] The target cell may be a cancer cell, or a cell capable of presenting a
peptide
from an infectious agent on an MHC class receptor. The target cell may or may
not
express a tumour-specific antigen, for example a dysfunctional P2X7 receptor.
[0023] In any aspect, the target cell may be any cell expressing a
dysfunctional P2X7
receptor, for example a cancer cell.
[0024] In any embodiment, 2 or more bridging molecules may be administered to
a
subject, each bridging molecule comprising a targeting moiety that binds to a
different
cell surface molecule on a target cell. For example, in the context of a
method of
treating cancer, each bridging molecule administered may comprise different
targeting
moieties and may therefore bind to a different tumour associated antigen
present on the
cancer cells. Such embodiments facilitate redirection of a single class of CAR
T cell to
multiple antigens present on tumour antigens (including at the same time) and
therefore
provide a multi-pronged approach for killing of cancer cells.
[0025] Accordingly, in any embodiment, the method of treating cancer comprises

administering 2 or more bridging molecules, wherein each bridging molecule
comprises
targeting moieties for binding to different cell surface antigens on a target
cell.
[0026] In further embodiments, the bridging molecules may bind to different
epitopes
on the same cell surface antigen expressed by the cancer cell. Accordingly, in
further
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embodiments, the methods of the invention comprise treating cancer comprises
administering 2 or more bridging molecules, wherein each bridging molecule
comprises
targeting moieties for binding to different epitopes on the same cell surface
antigen on a
target cell.
[0027] Further still, the invention provides for methods wherein bridging
molecules for
redirecting a CAR T cell to different cancer antigens, can be administered
synchronously to a subject in need thereof. This allows for fine-tuning of the
therapeutic
approach, such that a CAR T cell may be directed to binding cancer cells via
different
antigens, at different times during the course of the patient's therapeutic
regimen.
[0028] In further embodiments, a single bridging molecule may comprise more
than
one targeting moiety, such that a single molecule comprises targeting moieties
for more
than one cell surface molecule on a target cell.
[0029] Further still, a single bridging molecule may comprise more than one
targeting
moiety, such that a single molecule comprises targeting moieties for the same
cell
surface molecule on a target cell, but wherein the targeting moieties bind to
different
epitopes on the cell surface molecule.
[0030] In any embodiment of aspects directed to methods of treatment herein,
the
bridging molecule may be delivered via infusion to the subject or may be
expressed by
the immune cell expressing the chimeric antigen receptor. The bridging
molecule may
be a polypeptide, which is encoded in an inducible or a constitutive
expression construct
contained in the immune cell.
[0031] In any embodiment, the antigen-recognition domain binds to an epitope
associated with an adenosine triphosphate (ATP)-binding site of the
dysfunctional P2X7
receptor. In some embodiments, the dysfunctional P2X7 receptor has a reduced
capacity to bind ATP at the ATP-binding site compared to an ATP-binding
capacity of a
functional P2X7 receptor (e.g., a receptor having wild-type sequence and
having a
conformation or fold of an ATP-binding receptor). In some embodiments the
dysfunctional P2X7 receptor cannot bind ATP at the ATP-binding site.
[0032] In any embodiment, the dysfunctional P2X7 receptor has a conformational

change that renders the receptor dysfunctional. In some embodiments, the
conformational change is a change of an amino acid from the trans-conformation
to the
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cis-conformation. In some embodiments, the amino acid that has changed from a
trans-
conformation to a cis-conformation is proline at amino acid position 210 of
the
dysfunctional P2X7 receptor.
[0033] In any embodiment, the antigen-recognition domain binds to an epitope
that
includes the proline at amino acid position 210 of the dysfunctional P2X7
receptor. In
some embodiments, the antigen-recognition domain binds to an epitope that
includes
one or more amino acid residues spanning from glycine at amino acid position
200 to
cysteine at amino acid position 216, inclusive, of the dysfunctional P2X7
receptor.
[0034] The antigen-recognition domain of the receptor can be any suitable
molecule
that can interact with and specifically binds to a dysfunctional P2X7
receptor. However,
in some embodiments, the antigen-recognition domain includes amino acid
sequence
homology to the amino acid sequence of an antibody, or a fragment thereof,
which
binds to the dysfunctional P2X7 receptor. In some embodiments, the antigen-
recognition
domain includes amino acid sequence homology to the amino acid sequence of a
fragment-antigen binding (Fab) portion of an antibody that binds to a
dysfunctional P2X7
receptor. In some embodiments, the antibody is a humanised antibody.
[0035] In any embodiment, the antigen-recognition domain includes amino acid
sequence homology to the amino acid sequence of a single-chain variable
fragment
(scFv) or a multivalent scFv that binds to a dysfunctional P2X7 receptor. In
some
embodiments, the multivalent scFv is a divalent or trivalent scFv.
[0036] In any embodiment, the antigen-recognition domain includes amino acid
sequence homology to a single-antibody domain (sdAb) that binds to a
dysfunctional
P2X7 receptor.
[0037] In any embodiment, the antigen-recognition domain includes a binding
polypeptide that includes amino acid sequence homology to one or more
complementarity determining regions (CDRs) of an antibody that binds to a
dysfunctional P2X7 receptor. In any embodiment, the binding polypeptide
includes
amino acid sequence homology to the CDR1, 2 and 3 domains of the VH and/or VL
chain of an antibody that binds to a dysfunctional P2X7 receptor. In preferred

embodiments, the binding polypeptide comprises the amino acid sequence of the
CDRs
of the VH and/or VL chain of an antibody, or the amino acid sequence of the VH
and/or
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VL chains of an antibody, or the amino acid sequence of an antibody or
fragment
thereof, wherein the antibody or fragment thereof comprises the amino acid
sequences
of any antibody described in PCT/AU2002/000061 or PCT/AU2002/001204 (or in any

one of the corresponding US patents US 7,326,415, US 7,888,473, US 7,531,171,
US
8,080,635, US 8,399,617, US 8,709,425, US 9,663,584, or US 10,450,380),
PCT/AU2007/001540 (or in corresponding US patent US 8,067,550),
PCT/AU2007/001541 (or in corresponding US publication US 2010-0036101),
PCT/AU2008/001364 (or in any one of the corresponding US patents US 8,440,186,
US
9,181,320, US 9,944,701 or US 10,597,451), PCT/AU2008/001365 (or in any one of
the
corresponding US patents US 8,293,491 or US 8,658,385), PCT/AU2009/000869 (or
in
any one of the corresponding US patents US 8,597,643, US 9,328,155 or US
10,238,716), PCT/AU2010/001070 (or in any one of the corresponding
publications
VVO/2011/020155, US 9,127,059, US 9,688,771, or US 10,053,508), and
PCT/AU2010/001741 (or in any one of the corresponding publications WO
2011/075789
or US 8,835,609) the entire contents of which are hereby incorporated by
reference.
Preferably the antibody comprises the CDR amino acid sequences of 2-2-1
described in
PCT/AU2010/001070 (or in any one of the corresponding US patents US 9,127,059,
US
9,688,771, or US 10,053,508) or BPM09 described in PCT/AU2007/001541 (or in
corresponding US publication US 2010-0036101) and produced by the hybridoma
AB253 deposited with the European Collection of Cultures (ECACC) under
Accession
no. 06080101.
[0038] In some embodiments, the signalling domain includes a portion derived
from
an activation receptor. In some embodiments, the activation receptor is a
member of the
CD3 co-receptor complex or is an Fe receptor. In some embodiments, the portion

derived from the CD3 co-receptor complex is CD3-. In some embodiments, the
portion
derived from the Fc receptor is FoeRI or FcyRI.
[0039] In some embodiments, the signalling domain includes a portion derived
from a
co-stimulatory receptor. In some embodiments, the signalling domain includes a
portion
derived from an activation receptor and a portion derived from a co-
stimulatory receptor
In some embodiments, the co-stimulatory receptor is selected from the group
consisting
of CD27, CD28, CD30, CD40, DAP10, 0X40, 4-1BB (CD137) and !COS.
[0040]
In any aspect, the cell expressing an antigen receptor may be an immune
cell,
for example an immune cell as described herein, or a cell that is capable of
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differentiating into an immune cell (e.g., a progenitor of an immune cell). A
cell that is
capable of differentiating into an immune cell (e.g. T cell that will express
the
dysfunctional P2X7 CAR) may be a stem cell, multi-lineage progenitor cell or
induced
pluripotent stem cell.
[0041] In any embodiment of the aforementioned aspects, the receptor
comprising an
antigen-recognition domain and a signalling domain is a chimeric receptor
antigen
(CAR).
[0042] In any aspect, the targeting moiety that binds to a cell surface
molecule on a
target cell comprises or consists of a peptide or antibody or antibody
fragment.
Alternatively, the targeting moiety may comprise a ligand or binding partner
for a protein
or receptor present on the target cell surface.
[0043] The targeting moiety may further comprise a soluble T cell receptor
(TcR) or a
single chain T cell receptor binding motif or a T cell receptor-like mAb. In
such
embodiments, the targeting moiety is particularly suitable for the binding of
peptides
derived from intracellularly processed proteins from infectious agents that
are presented
on a cell surface via MHC (HLA) I and ll molecules. The targeting moiety may
also be
suitable for binding of peptides presented by MHC molecules, wherein the
peptides
comprise mutations associated with cancers, such as the cancer testis antigens
(WTI,
NY-ESO-1, PRAME family (e.g. PRA100, PRA142, PRA300, PRA425 and others),
MAGE family (e.g., MAGE-Al, MAGE-A3, MAGE-A4, MAGE-Al2 and others), CT83,
SSX2, GAGE, BAGE, PAGE) or other cancer specific mutations.
[0044] In any embodiment, the cell surface molecule may comprise an antigen,
preferably an antigen as described herein.
[0045] The cell surface molecule may be selected from a protein, a lipid
moiety, a
glycoprotein, a glycolipid, a carbohydrate, a polysaccharide, a nucleic acid,
an MHC-
bound peptide, or a combination thereof.
[0046] The cell surface molecule may comprise parts (e.g., coats, capsules,
cell
walls, flagella, fimbrae, and toxins) of bacteria, viruses, and other
microorganisms. The
cell surface molecule may be expressed by the target cell.
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[0047] The cell surface molecule may not be expressed by the target cell. By
way of
non-limiting example, the cell surface molecule may be a ligand expressed by a
cell that
is not the target cell and that is bound to the target cell or a cell surface
molecule of the
target cell. Also, by non-limiting example, the cell surface molecule may be a
toxin,
exogenous molecule or viral protein that is bound to a cell surface or cell
surface
receptor of the target cell.
[0048] In any aspect or embodiment, the targeting moiety of the bridging
molecule
does not bind to the same antigen or epitope as the antigen-recognition of the
receptor.
For example, the targeting moiety of the bridging molecule does not bind to a
dysfunctional P2X7 receptor, the E200, E300, or E200/E300 composite epitope,
or any
other epitope present on a dysfunctional P2X7 receptor as described herein.
[0049] The targeting moiety may be a targeting antibody or antibody fragment.
The
targeting antibody or antibody fragment may be an immunoglobulin (Ig). The
immunoglobulin may be selected from an IgG, an IgA, an IgD, an IgE, an IgM, a
fragment thereof or a modification thereof. The immunoglobulin may be IgG. The
IgG
may be IgG1. The IgG may be any IgG subclass.
[0050] In any embodiment, a bridging molecule of the invention may comprise
more
than one targeting moiety. For example, in certain non-limiting embodiments,
the
bridging molecule may comprise two different antibodies, or fragment thereof.
The
antibodies may bind different epitopes of the same cell surface molecule on
the target
cell. Alternatively, the antibodies may bind epitopes of different cell
surface molecules
on the target cell.
[0051] The dysfunctional P2X7 receptor epitope moiety may be provided in the
form
of a P2X7 receptor, or a fragment of a P2X7 receptor that has at least one of
the three
ATP binding sites that are formed at the interface between adjacent correctly
packed
monomers that are unable to bind ATP. Such receptors are unable to extend the
opening of the non-selective calcium channels to apoptotic pores.
[0052]
In any aspect, the dysfunctional P2X7 receptor epitope moiety comprises or
consists of a fragment of a dysfunctional P2X7 receptor. Exemplary fragments
include
GHNYTTRNILPGLNITC (SEQ ID NO: 2; also referred to herein as the "E200
epitope")
and variants thereof (exemplary variants are provided in SEQ ID NOs: 3 to 10
and 15 to
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30 and 168); KYYKENNVEKRTLIKVF (SEQ ID NO: 12 and 13; also referred to herein
as the "E300" epitope); or GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 14; also
referred to herein as the "E200/E300" or "cornposite" epitope). Further
examples are
provided in Table 1 herein.
[0053] In any aspect, the dysfunctional P2X7 receptor epitope moiety is bound
by an
antibody that binds to dysfunctional P2X7 receptors, but is not bound by
antibodies that
bind to functional P2X7 receptors.
[0054] In any aspect, a bridging molecule may comprise 2 or more dysfunctional

P2X7 receptor epitope moieties. The 2 or more dysfunctional P2X7 receptor
epitope
moieties may comprise or consist of the same sequence, or of different
sequences. For
example, in any aspect, a bridging molecule may comprise a dysfunctional P2X7
receptor epitope moiety in the form of the E200 epitope and a further
dysfunctional
P2X7 receptor epitope moiety in the form of the E300 epitope. Alternatively,
in any
aspect, a bridging molecule may comprise a dysfunctional P2X7 receptor epitope
moiety
in the form of the E200 epitope and a further dysfunctional P2X7 receptor
epitope
moiety in the form of the composite epitope. Still further, in any aspect, a
bridging
molecule may comprise a first dysfunctional P2X7 receptor epitope moiety in
the form of
the E200 epitope and a further dysfunctional P2X7 receptor epitope moiety in
the form
of the E200 epitope.
[0055] As used herein, except where the context requires otherwise, the term
"comprise" and variations of the term, such as "comprising", "comprises" and
"comprised", are not intended to exclude further additives, components,
integers or
steps.
[0056] Further aspects of the present invention and further embodiments of the

aspects described in the preceding paragraphs will become apparent from the
following
description, given by way of example and with reference to the accompanying
drawings.
Brief description of the drawings
[0057] Figure 1: Diagrammatic representation of exemplary bridging molecules.
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[0058] Figure 2: Diagrammatic representation of exemplary embodiments of the
present invention utilising nfP2X7-CAR and Fab-based E200 bridging molecules
targeting cancer, infection and immunomodulatory-related target antigens.
[0059] Figure 3: Diagrammatic representation of exemplary embodiments of the
present invention utilising nfP2X7-CAR and single chain TCR-based E200
bridging
molecules targeting cancer and infection-related peptides.
[0060] Figure 4: Bridging molecules in Fab format with a single E200 epitope
either
directly linked to the VH ((a) and (b)) or via a linker ((c) and (d)) binds to
CD19 on JeKo-
1 (mantle cell lymphoma) cell line and the E200 epitope is available for
binding to an
antibody (BIL03_2-2-1 ¨ AF647). HIS tag is detected by FITC antibody. (a) and
(c) show
anti-HIS antibody binding, (b) and (d) show binding of antibody to
dysfunctional P2X7
receptor epitope.
[0061] Figure 5: Bridging molecules in scFv format with a single E200 epitope
either
directly linked to the VH ((a) and (b)) or via a linker ((c) and (d)) binds to
CD19 on JeKo-
1 (mantle cell lymphoma) cell line and the E200 epitope is available for
binding to an
antibody. (a) and (c) show anti-HIS antibody binding, (b) and (d) show binding
of
antibody to dysfunctional P2X7 receptor epitope.
[0062] Figure 6: Bridging molecules in Fab format with a single E200 epitope
either
directly linked to the VL ((a) and (b)) or via a linker ((c) and (d)) binds to
CD19 on JeKo-
1 (mantle cell lymphoma) cell line and the E200 epitope is available for
binding to an
antibody. (a) and (c) show anti-HIS antibody binding, (b) and (d) show binding
of
antibody to dysfunctional P2X7 receptor epitope.
[0063] Figure 7: Bridging molecules in scFv format with a single E200 epitope
either
directly linked to the VL ((a) and (b)) or via a linker ((c) and (d)) binds to
CD19 on JeKo-
1 (mantle cell lymphoma) cell line and the E200 epitope is available for
binding to an
antibody. (a) and (c) show anti-HIS antibody binding, (b) and (d) show binding
of
antibody to dysfunctional P2X7 receptor epitope.
[0064] Figure 8: Binding of bridging molecules to various antigens 0D37,
CD79B,
ROR1, CD33, CD38, CD123, CD135, BCMA, EGFR, PDL1, CD22, CD70 and CD20.
(a), (c), (e), (g), (i), (k), (m), (o), (q), (s), (u), (w) and (y) show anti-
HIS antibody binding,
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(b), (d), (f), (h), (j), (I), (n), (p), (r), (t), (v), (x) and (z) show
binding of antibody to
dysfunctional P2X7 receptor epitope.
[0065] Figure 9: "painting" of JeKo-1 cells with CD19 targeted Fab bridging
molecules in the illustrated format as detected by flow cytometry. Cells were
incubated
at indicated concentrations with Fab bridging molecules. CD33 targeted Fab
bridging
molecules served as negative control in JeKo-1 at 10 ng/mL and 1000 ng/mL.
C019
targeted Fab bridging molecules were used at 1 ng/mL, 10 ng/mL, 100 ng/mL and
1000
ng/mL.
[0066] Figure 10: "painting" of MOLM-13 cells with 0033 targeted Fab bridging
molecules in the illustrated format as detected by flow cytometry. Cells were
incubated
at indicated concentrations with Fab bridging molecules. CD19 targeted Fab
bridging
molecules served as negative control in JeKo-1 at 10 ng/mL and 1000 ng/mL.
0D33
targeted Fab bridging molecules were used at 1 ng/mL, 10 ng/mL, 100 ng/mL and
1000
ng/mL.
[0067] Figure 11: Illustrates the "painting" of MOLM-13 (AML) cells via 0033
targeted Fab bridging molecules. The flow data shows in black the isotype
control, in
blue the staining with BIL03 2-2-1 sd-mAb only at 1 ug/mL and in green the
increase of
staining via the combination of C033 targeted Fab bridging molecules and BIL03
2-2-1
sd-mAb. The increase of target molecules that can be recognised by the nfP2X7
BRIDGE CAR expressing effector cells translate into CAR-mediated effector
function.
The use of the bridging molecules enhances the CAR function by increasing the
targeting epitopes on the cancer cells.
[0068]
Figure 12: A representative flow cytometric plot with direct comparison of
untransduced T cells (left panel), CAR0007_hPGK (middle panel) and
CAR0007_EF1a
(right panel) expressing T cells. Both CAR T cells containing conditions
showed
significantly increased expression of the activation markers 0025 and 0D69 in
incubation with MOLM-13 at 20:1 ET ratio and C033 targeted Fab bridging
molecules at
1000 ng/mL after 48 hours.
[0069] Figure 13: The flow cytometric plots of T cells and MOLM-13 at 20:1 ET
ratio
and C033 targeted Fab bridging molecules at 1000 ng/mL after 48 hours
corresponding
to Figure 12 showed a complete clearance of leukaemic cells in the conditions
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containing CAR0007_hPGK and CAR0007_EF1a whereas there was no relevant
impact on leukaemic cell number in the untransduced T cell condition.
[0070] Figure 14: Flow cytometric plots that illustrate the dose dependent
clearance
of leukaemic cells at indicated CD33 targeted Fab bridging molecule
concentrations of
CAR0007_EF1a containing T cells and MOLM-13 at 20:1 ET ratio after 48 hours.
As
low as 40 ng/mL showed almost complete elimination of leukaemic cells and
complete
elimination of leukaemic cells at 200 and 1000 ng/mL.
[0071] Figure 15: (a) Specific lysis of MOLM-13 leukaemic cells by
CAR0007_hPGK
T cells at an ET ratio of 20:1 after 48 hour incubation with and without EGFR
and 0D33
targeted bridging molecules at indicated concentrations is illustrated.
Significant lysis in
a dose dependent manner was found for increasing concentrations (40, 200 and
1000
ng/mL) of 0D33 targeted Fab bridging molecules. (b) Specific lysis of MOLM-13
leukaemic cells by CAR0007_hEF1a T cells at an ET ratio of 20:1 after 48 hour
incubation with and without EGFR and 0D33 targeted bridging molecules at
indicated
concentrations is illustrated. Significant lysis in a dose dependent manner
was found for
increasing concentrations (40, 200 and 1000 ng/mL) of CD33 targeted Fab
bridging
molecules.
[0072] Figure 16: A titration experiment of EGFR and CD33 targeted Fab-
bridging
molecules was used to test the impact on killing of MOLM-13 by CAR0007_hPGK.
There was a significant impact on the viability of MOLM-13 after 24 hour
incubation at
10:1 ET ratio between the condition with EGFR targeted bridging molecules and
CD33
targeted bridging molecules at 1000 ng/mL. Statistical analysis was performed
by t-test.
[0073] Figure 17: Alternative representation of the data from Figure 16. There
was
no significant difference in the titration of the EGFR bridging molecules
(data not
shown). There were significant differences in the titration of the CD33
bridging
molecules. Statistical analyses were performed by One-Way ANOVA and post-hoc
test
Tukey.
[0074] Figure 18: A titration experiment of EGFR and 0D33 targeted Fab-
bridging
molecules was used to test the impact on killing of MOLM-13 by CAR0007_hPGK.
There was a significant impact on the viability of MOLM-13 after 24 hour
incubation at
20:1 ET ratio between the condition with EGFR targeted bridging molecules and
0033
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targeted bridging molecules at 200 ng/mL and 1000 ng/mL. Statistical analysis
was
performed by t-test.
[0075] Figure 19: Alternative representation of the data from Figure 18. There
was
no significant difference in the titration of the EGFR bridging molecules
(data not
shown). There were significant differences in the titration of the CD33
bridging
molecules. Statistical analyses were performed by One-Way ANOVA and post-hoc
test
Tukey.
[0076] Figure 20: A titration experiment of EGFR and CD33 targeted Fab-
bridging
molecules was used to test the impact on killing of MOLM-13 by CAR0007_hPGK.
There was a significant impact on the viability of MOLM-13 after 48 hour
incubation at
10:1 ET ratio between the condition with EGFR targeted bridging molecules and
CD33
targeted bridging molecules at 200 ng/mL and 1000 ng/mL. Statistical analysis
was
performed by t-test.
[0077] Figure 21: A titration experiment of EGFR and 0D33 targeted Fab-
bridging
molecules was used to test the impact on killing of MOLM-13 by CAR0007_hPGK.
There was a significant impact on the viability of MOLM-13 after 48 hour
incubation at
20:1 ET ratio between the condition with EGFR targeted bridging molecules and
C033
targeted bridging molecules at 200 ng/mL and 1000 ng/mL. Statistical analysis
was
performed by t-test.
[0078] Figure 22: A titration experiment of EGFR and CD33 targeted Fab-
bridging
molecules was used to test the impact on killing of MOLM-13 by CAR0007_EF1a.
There
was a significant impact on the viability of MOLM-13 after 24 hour incubation
at 10:1 ET
ratio between the condition with EGFR targeted bridging molecules and CD33
targeted
bridging molecules at 200 ng/mL and 1000 ng/mL. Statistical analysis was
performed by
t-test.
[0079] Figure 23: Alternative representation of the data from Figure 22. There
was
no significant difference in the titration of the EGFR bridging molecules
(data not
shown). There were significant differences in the titration of the 0D33
bridging
molecules. Statistical analyses were performed by One-Way ANOVA and post-hoc
test
Tukey.
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[0080] Figure 24: A titration experiment of EGFR and CD33 targeted Fab-
bridging
molecules was used to test the impact on killing of MOLM-13 by CAR0007_EF1a.
There
was a significant impact on the viability of MOLM-13 after 24 hour incubation
at 20:1 ET
ratio between the condition with EGFR targeted bridging molecules and CD33
targeted
bridging molecules at 40 ng/mL, 200 ng/mL and 1000 ng/mL. Statistical analysis
was
performed by t-test.
[0081] Figure 25: Alternative representation of the data from Figure 24. There
was
no significant difference in the titration of the EGFR bridging molecules
(data not
shown). There were significant differences in the titration of the CD33
bridging
molecules. Statistical analyses were performed by One-Way ANOVA and post-hoc
test
Tukey.
[0082] Figure 26: A titration experiment of EGFR and 0D33 targeted Fab-
bridging
molecules was used to test the impact on killing of MOLM-13 by CAR0007_EF1a.
There
was a significant impact on the viability of MOLM-13 after 48 hour incubation
at 10:1 ET
ratio between the condition with EGFR targeted bridging molecules and CD33
targeted
bridging molecules at 40 ng/mL, 200 ng/mL and 1000 ng/mL. Statistical analysis
was
performed by t-test.
[0083] Figure 27: A titration experiment of EGFR and CD33 targeted Fab-
bridging
molecules was used to test the impact on killing of MOLM-13 by CAR0007_EF1a.
There
was a significant impact on the viability of MOLM-13 after 48 hour incubation
at 20:1 ET
ratio between the condition with EGFR targeted bridging molecules and CD33
targeted
bridging molecules at 40 ng/mL, 200 ng/mL and 1000 ng/mL. Statistical analysis
was
performed by t-test.
[0084] Figure 28: (a) A kill assay (cytotoxicity was measure by quantification
of
residual leukaemic cells compared with a control) the elimination of leukaemic
cells at
the EGFR bridging molecule concentrations 40, 200 and 1000 ng/mL showed no
significant difference between untransduced T cells compared with CAR T cells.
(b)
The elimination of leukaemic cells at the CD33 bridging molecule
concentrations 40,
200 and 1000 ng/mL showed a significant difference between untransduced T
cells
compared with both CAR0007 transduced T cells (hPGK and EF1a).
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[0085] Figure 29: A titration experiment of CD33 targeted Fab-bridging
molecules
was used to test the impact on killing of MOLM-13 by untransduced T cells,
CAR0007_hPGK and CAR0007_EF1a effector cells at an ET ratio 10:1 after 48 hour

incubation.
[0086] Figure 30: Analysis of the results in Figure 29. There was a consistent

significant difference in the titration of the C033 bridging molecules between
the three
effector cell populations at 40 ng/mL, 200 ng/mL and 1000 ng/mL. Statistical
analyses
were performed by One-Way ANOVA and post-hoc test Tukey to compare the named
three conditions per concentration of EGFR bridging molecules.
[0087] Figure 31: A titration experiment of CD33 targeted Fab-bridging
molecules
was used to test the impact on killing of MOLM-13 by untransduced T cells,
CAR0007_hPGK and CAR0007_EF1a effector cells at an ET ratio 20:1 after 48 hour

incubation
[0088] Figure 32: Analysis of the results in Figure 31. There was a consistent

significant difference in the titration of the CD33 bridging molecules between
the three
effector cell populations at 8 ng/mL, 40 ng/mL, 200 ng/mL and 1000 ng/mL.
Statistical
analyses were performed by One-Way ANOVA and post-hoc test Tukey to compare
the
named three conditions per concentration of EGFR bridging molecules.
[0089] Figure 33: Cell killing by nfP2X7 CAR-T cells in the presence of CD19-
targeting bridging molecules in Fab and IgG1 format and with different
dysfunctional
P2X7 receptor epitope moieties. CAR10 versus JeKo-1 Effector cell / Target
cell (ET)
ratio 10:1. CAR10 to target 2.9:1. N= 1 healthy donor. 6 replicates. CAR10
expression
(d12) = 28.6%. Indicated significance is the group versus the control (****)
are all
significantly different from the control.
[0090] Figure 34: Cell killing by nfP2X7 CAR-T cells in the presence of CD19-
targeting bridging molecules with different dysfunctional P2X7 receptor
epitope moieties.
CAR10 versus JeKo-1 Effector cell / Target cell (ET) ratio 20:1. N= 1 healthy
donor. 100
ng/mL Fab CD19 bridging molecules. 6 replicates. In all CAR conditions, CAR
expressing cells were normalised to ET ratio 1.48:1. Indicated significance is
the group
versus the control (****) are all significantly different from the control.
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[0091] Figure 35: Cell killing by three different classes of nfP2X7 CAR-T
cells (i.e.
having different CAR architectures), in the presence of C1J19-targeting
bridging
molecules in Fab and IgG1 format. CAR7, CAR10 and CAR16 versus JeKo-1 Effector

cell / Target cell (ET) ratio 10:1. CAR7 to target 3.3:1; CAR10 to target
2.8:1. CAR16 to
target 2.2:1. N= 1 healthy donor. 6 replicates. CAR7 expression (d12) 33.3%;
CAR10
expression (d12) = 28.6%; CAR16 expression (d12) = 22.6%. Indicated
significance is
the group versus the control (****) are all significantly different from the
control.
[0092] Figure 36: Cell killing by nfP2X7 CAR-T cells in the presence of A)
CD19-
targeting bridging molecules in Fab format. (killing of JeKo-1 cells) or b)
CD33-targeting
bridging molecule in Fab format (killing of MOLM-13 cells).
[0093] Figure 37: Schematic of in vivo evaluation of bridging molecules.
[0094] Figure 38: bioluminescence (total Flux, p/s) as an indicator of tumour
burden
in mice following inoculation of Jeko-1_LUC_eGFP cells. Mice received
treatment with:
Group 1 = no treatment; Group 2 = activated untransduced T cells; Group 3 =
bridging
molecule (E200 epitope + anti-CD19 Fab); Group 4 = activated untransduced T
cells +
bridging molecule; Group 5 = 3rd generation anti-CD19 CAR T cells; Group 6 = T
cells
expressing anti-nfP2X7 receptor CAR 1; Group 7 = CAR 1-T cells + bridging
molecule;
Group 8 - T cells expressing nfP2X7 receptor CAR 2; Group 9 = CAR 2-T cells +
bridging molecule.
Table 1: Sequence information
Description Sequence
SEO ID NO:
Exemplary dysfunctional P2X7 receptor epitope moiety sequences
Human P2X7 M PACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVI I FSYVCFALV
1
receptor SDKLYQRKEPVISSVHTKVKGIAEVKE [IV ENGVKKLVHSVF DTAD
YTFPLQGNSFFVMTN FLKTEGQEQRLCPEYPTRRTLCSSDRGCK
KGWMDPQSKGIQTG RCVVYEGNQKTCEVSAWCPIEAVEEAPRP
ALLNSAENFTVLIKNN I DFPG HNYTTRN I LPG LN ITCTFHKTQN PQC
PI FRLGDIFR ETGDNFSDVAIQGGIMGI E IYWDCNLDRWFHHCR PK
YSFR RLDDKTTNVSLYPGYN FRYAKYYKENNV EKRTLI KV FG I RFD
I LVFGTGGKFDI IQLVVYIGSTLSYFGLAAVFIDFLI DTYSSNCCRSHI
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YPWCKCCQPCVVNEYYYRKKCESIVEPKPTLKYVSFVDESH IRM
VNQQLLGRSLQDVKGQEVPRPAMDFTDLSRLPLALHDTPPIPGQ
PEEIQLLRKEATPRSRDSPVVVCQCGSCLPSQLPESHRCLEELCC
RKKPGACITTSELFRKLVLSRHVLQFLLLYQEPLLALDVDSTNSRL
RHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQY
SGFKSPY
Exemplary E200 GHNYTTRNILPGLNITC
2
epitope
Variant E200 GHNYTTRNILPGLNIT 3
epitope peptide
(E200')
E200 epitope Cys GHNYTTRNILPGLNITS
4
to Ser
modification
Extended E200 GHNYTTRNILPGLNITSTFHK
5
Cys to Ser
modification
Extended E200' GHNYTTRNILPGLNITSTFHKT
6
Cys to Ser
modification
(22 aa)
Extended E200" GHNYTTRNILPGLNITSTFHKTC
7
Cys to Ser
modification
Pep16 DFPGHNYTTRNILPGC
8
Pep17 GHNYTTRNILPGLNITSTFHKTS
9
Extended Pep17 GHNYTTRNILPGLNITSTFHKTSGSGK
10
(27 aa)
Minimum NYTTRNILPGL
11
sequence E200
peptide (target
epitope)
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Exemplary E300 KYYKENNVEKRTLIK
12
epitope
Variant E300 KYYKENNVEKRTLIKVF
13
epitope peptide
(E30')
Exemplary GHNYTTRNILPGAGAKYYKENNVEK
14
E200/E300 or
composite epitope
E200 + G46 GHNYTTRNILPGLNITSGGGGS
15
lin ker
E200 + 2xG4S GHNYTTRNILPGLNITSGGGGSGGGGS
16
lin ker
E200+3xG4S GHNYTTRNILPGLNITSGGGGSGGGGSGGGGS
168
lin ker
E200_extended GHNYTTRNILPGLNITSTFHKTGS
17
peptide 17v3 (24
aa)
E200_extended GHNYTTRNILPGLNITSTFHGS
18
peptide 17v4 (22
aa)
E200_extended GHNYTTRNILPGLNITSGS
19
peptide 17v5 (19
aa)
E200_extended DFPGHNYTTRNILPGLNITSGS
20
peptide 17v6 (22
aa)
(E200 epitope
extended into N
terminal region)
E200_extended DFPGHNYTTRNILPGLNITSGGGGS
21
peptide 17v7 (25
aa) + linker
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E200_extended DFPGHNYTTRNILPGLNITSGGGGSGGGGS
22
peptide 17v8 (30
aa) + linker
E200_extended DFPGHNYTTRNILPGLNITSGGGGSGGGGSGGGGS
23
peptide 17v9 (35
aa) + linker
E200_extended DFPGHNYTTRNILPGLNITSTFHKTSGSGK
24
peptide 17v10 (30
aa)
E200_extended DFPGHNYTTRNILPGLNITSTFHKTSGSGKGS
25
peptide 17v11 (32
aa) + linker
E200_extended DFPGHNYTTRNILPGLNITSTFHKTSGSGKGGGGS
26
peptide 17v12 (35
aa) + linker
E200_extended DFPGHNYTTRNILPGLNITSTFHGGGGS
27
peptide 17v13 (25
aa) + linker
E200_extended GHNYTTRNILPGLNITSTFHGGGGS
28
peptide 17v14 (22
aa) + linker
E200_extended DFPGHNYTTRNILPGLNITSTFHKTGGGGS
29
peptide 17v15 (30
aa) + linker
E200_extended GHNYTTRNILPGLNITSTFHKTGGGGS
30
peptide 17v16 (27
aa) +
Exemplary targeting moiety sequences and exemplary bridging molecules
Constructs based on FMC63 (for binding CD19)
CD19 binder EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSVVIRQPPRKG
31
heavy chain LEVVLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDT
AIYYCAKHYYYGGSYAMDYVVGQGTSVTVSSASTKGPSVFPLAPS
CD19, FMC63,
SKSTSGGTAALGCLVKDYFPEPVTVSINNSGALTSGVHTFPAVLQ
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B001_Heavy SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
Chain HHHHHH
CD19 binder light DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNVVYQQKPDGTVK
32
chain LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG
NTLPYTFGGGTKLEITKARTVAAPSVFIFPPSDEQLKSGTASVVCL
CD19, FMC63, LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
B001_Light Chain
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
E200+CD19 GHNYTTRNILPGLNITSEVKLQESGPGLVAPSQSLSVTCTVSGVSL 33
binder (nfP2X7 PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNS
epitope KSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYINGQGTSVT
underlined) VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
CD19, FMC63,
HKPSNTKVDKKVEPKSCHHHHHH
B002-1_Heavy
Chain
E200+CD19 GHNYTTRNILPGLNITSGGGGSEVKLQESGPGLVAPSQSLSVTCT 34
binder (nfP2X7 VSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL
epitope TI I KD NSKSQVFLKMNSLQTDDTA IYYCAKHYYYGGSYAM DYVVG
underlined) QGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
CD19, FMC63,
YICNVNHKPSNTKVDKKVEPKSCHHHHHH
B002-2_Heavy
Chain
E200+CD19 GHNYTTRNILPGLNITSDIQMTQTTSSLSASLGDRVTISCRASQDIS 35
binder (nfP2X7 KYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTI
epitope SNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKARTVAAPSVFIFP
underlined) PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
CD19, FMC63,
SFNRGEC
Light chain B003-
1
E200+CD19 GHNYTTRNILPGLNITSGGGGSDIQMTQTTSSLSASLGDRVTISCR 36
binder (nfP2X7 ASQDISKYLNVVYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG
epitope TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKARTVAA
underlined) PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
CD19, FMC63, LSSPVTKSFNRGEC
Light chain B003-
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2
E2004CD19 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSVVIRCIPPRKG 37
binder (nfP2X7
LEVVLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDT
epitope
AIYYCAKHYYYGGSYAMDYVVGQGTSVTVSSASTKGPSVFPLAPS
underlined) SKSTSGGTAALGCLVKDYFPEPVTVSINNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
CD19, FMC63,
GHNYTTRNILPGLNITSHHHHHH
B005_Heavy
Chain
CD19, FMC63, DIQMTOTTSSLSASLGDRVTISCRASCIDISKYLNVVYQQKPDGTVK 38
B005_Light Chain LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG
NTLPYTFGGGTKLEITKARTVAAPSVFIFPPSDEQLKSGTASVVCL
(His tagged
LNNFYPREAKVQVVKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
version of SEQ ID
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECHHHHHH
NO: 32)
E200+CD19 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNVVYQQKPDGTVK 39
binder (nfP2X7
LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG
epitope
NTLPYTFGGGTKLEITKARTVAAPSVFIFPPSDEQLKSGTASVVCL
underlined)
LNNFYPREAKVQVVKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGHNYTTRNIL
CD19, FMC63, PG LN ITS
B006_Light Chain
CD19 binder scFv DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNVVYQQKPDGTVK 40
format LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG
NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLV
CD19, FMC63, APSQSLSVTCTVSGVSLPDYGVSVVIRQPPRKGLEVVLGVIWGSET
TYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG
B011_scFv_Light/
GSYAMDYVVGQGTSVTVSSHHHHHH
Heavy
CD19 binder scFv EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSVVIRQPPRKG 41
format LEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDT
AIYYCAKHYYYGGSYAMDYVVGQGTSVTVSSGGGGSGGGGSGG
CD19, FMC63,
GGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD
B012_scFv_Heav GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYF
y/Light
CQQGNTLPYTFGGGTKLEITHHHHHH
E200+CD19 GHNYTTRN ILPGLN ITSD
IQMTQTTSSLSASLGDRVTISCRASQD IS 42
binder in scFv
KYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTI
format (nfP2X7 SNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG
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epitope GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP
underlined) RKGLEWLGVIVVGSETTYYNSALKSRLTIIKONSKSQVFLKMNSLQ
TDDTAIYYCAKHYYYGGSYAMDYVVGQGTSVTVSSHHHHHH
CD19, FMC63,
B013-1_scFv_LH
E200+CD19 GHNYTTRNILPGLNITSGGGGSDIQMTQTTSSLSASLGDRVTISCR 43
binder in scFv ASQDISKYLNVVYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG
format (nfP2X7 TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSG
epitope GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV
underlined) SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFL
KMNSLQTDDTAIYYCAKHYYYGGSYAMDYVVGQGTSVTVSSHHH
CD19, FMC63, HHH
B013-2_scFv_LH
E200+CD19 GHNYTTRNILPGLNITSEVKLQESGPGLVAPSQSLSVTCTVSGVSL 44
binder in scFv PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNS
format (nfP2X7 KSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYVVGQGTSVT
epitope VSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRA
underlined) SQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT
DYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITHHHHHH
CD19, FMC63,
B014-1_scFv_HL
E200+CD19 GHNYTTRNILPGLNITSGGGGSEVKLQESGPGLVAPSQSLSVTCT 45
binder in scFv VSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL
format (nfP2X7 TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYVVG
epitope QGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRV
underlined) TISCRASQDISKYLNVVYQQKPDGTVKLLIYHTSRLHSGVPSRFSG
SGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITHHH
CD19, FMC63, HHH
B014-2_scFv_HL
E200+CD19 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSVVIRQPPRKG 46
binder in scFv LEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDT
format (nfP2X7 AIYYCAKHYYYGGSYAMDYVVGQGTSVTVSSGGGGSGGGGSGG
epitope GGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNVVYQQKPD
underlined) GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYF
CQQGNTLPYTFGGGTKLEITGHNYTTRN ILPGLN ITSHHHHHH
CD19, FMC63,
B015_scFv_HL
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E200+CD19 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVK 309
binder in scFv LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQG
format (nfP2X7 NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLV
epitope APSQSLSVTCTVSGVSLPDYGVSVVIRQPPRKGLEWLGVIWGSET
underlined) TYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG
GSYAMDYVVGQGTSVTVSSGHNYTTRNILPGLNITSHHHHHH
CD19, FMC63,
B016_scFv_LH
E200+CD19 GHNYTTRNILPGLNITSDIQMTQTTSSLSASLGDRVTISCRASQDIS 47
binder in scFv KYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTI
format (nfP2X7 SNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG
epitope GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP
underlined) RKGLEWLGVIVVGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ
TDDTAIYYCAKHYYYGGSYAMDYVVGQGTSVTVSSGHNYTTRNIL
CD19, FMC63,
PGLNITSHHHHHH
B017-1_scFv_LH
E200+CD19 GHNYTTRNILPGLNITSGGGGSDIQMTQTTSSLSASLGDRVTISCR 48
binder in scFv ASQDISKYLNVVYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSG
format (nfP2X7 TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSG
epitope GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGV
underlined) SWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFL
KMNSLQTDDTAIYYCAKHYYYGGSYAMDYVVGQGTSVTVSSGHN
CD19, FMC63, YTTRNILPGLNITSHHHHHH
B017-2_scFv_LH
E200+CD19 GHNYTTRNILPGLNITSEVKLQESGPGLVAPSQSLSVTCTVSGVSL 49
binder in scFv PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNS
format (nfP2X7 KSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYVVGQGTSVT
epitope VSSGGGGSGGGGSGGGGSDIQMTOTTSSLSASLGDRVTISCRA
underlined) SQDISKYLNVVYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT
DYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGHNYTTRN
CD19, FMC63, ILPGLNITSHHHHHH
B018-1_scFv_HL
E200+CD19 GHNYTTRNILPGLNITSGGGGSEVKLQESGPGLVAPSQSLSVTCT 50
binder in scFv VSGVSLPDYGVSWIRQPPRKGLEVVLGVIWGSETTYYNSALKSRL
format (nfP2X7 TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYVVG
epitope QGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRV
underlined) TISCRASQDISKYLNVVYQQKPDGTVKLLIYHTSRLHSGVPSRFSG
SGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGHN
CD19, FMC63,
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B018-2_scFv_HL YTTRNILPGLNITSHHHHHH
Constructs based on Tafasitamab (for binding CD19)
E200+CD19 GHNYTTRNILPGLNITSDIVMTQSPATLSLSPGERATLSCRSSKSL 51
binder (nfP2X7 QNVNGNTYLYVVFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGS
epitope GTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPS
underlined) VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
C019, PVTKSFNRGECHHHHHH
Tafasitamab,
B020-1_Light
Chain
CD19, EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMH\NVRQAPGK 52
Tafasitamab, GLEVVIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSE
B020-2_Heavy DTAMYYCARGTYYYGTRVFDYVVGQGTLVTVSSASTKGPSVFPLA
Chain PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LOSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPK
SCHHHHHH
Constructs for binding CD20
E200+CD20 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASSSV 53
binder (nfP2X7 SYMHVVYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTI
epitope SSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPP
underlined) SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
CD20,
FNRGECHHHHHH
Ocrelizumab,
B021-1_Light
chain
CD20, EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHVVVRQAPGK
54
Ocrelizumab, GLEVVVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLR
B021-1_Heavy AEDTAVYYCARVVYYSNSYVVYFDVWGQGTLVTVSSASTKGPSV
Chain FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCHHHHHH
E200+CD20 GHNYTTRNILPGLNITSEIVLTQSPATLSLSPGERATLSCRASQSV 55
binder (nfP2X7 SSYLAVVYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLT
epitope ISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPP
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underlined) SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
0020,
FNRGECHHHHHH
Ofatumumab,
B022-1_Light
Chain
CD20, EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHVVVRQAPGK
56
Ofatumumab, GLEVVVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRA
B022-2_Heavy EDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFP
Chain LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE
PKSCHHHHHH
Constructs for binding 0022
E200+CO22 GHNYTTRNILPGLNITSDIQMIQSPSSLSASVGDRVTITCRASQTIW 57
binder (nfP2X7 SYLNWYRQRPGEAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTI
epitope SSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKRTVAAPSVFIFPPS
underlined) DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
0022, m971-L7, NRGECHHHHHH
B023-1_LC
0D22, m971-L7, QVQLQQSGPGMVKPSQTLSLTCAISGDSVSSNSVAWNVVIRQSP
58
B023-2_Heavy SRGLEWLGRTYYRSTVVYNDYAVSMKSRITINPDTNKNQFSLQLN
Chain SVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSINNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCHHHHHH
E200+CD22 GHNYTTRNILPGLNITSDVQVTQSPSSLSASVGDRVTITCRSSQSL 59
binder (nfP2X7 ANSYGNTFLSWYLHKPGKAPQLLIYGISNRFSGVPDRFSGSGSGT
epitope DFTLTISSLQPEDFATYYCLQGTHQPYTFGQGTKVEIKRTVAAPSV
underlined) FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS
QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
0022, VTKSFNRGECHHHHHH
lnotuzumab,
B024-1_LC
CO22, EVQLVQSGAEVKKPGASVKVSCKASGYRFTNYVVIHWVRQAPGQ
60
lnotuzumab, GLEVVIGGINPGNNYATYRRKFQGRVTMTADTSTSTVYMELSSLR
B024-2_HC SEDTAVYYCTREGYGNYGAWFAYVVGQGTLVTVSSASTKGPSVF
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
29
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCHHHHHH
Constructs for binding CD79B
E200+CD79B GHNYTTRNILPGLNITSDIQLTQSPSSLSASVGDRVTITCKASQSV 61
binder (nfP2X7 DYEGDSFLNVVYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGT
epitope DFTLTISSLQPEDFATYYCQQSNEDPLTFGQGTKVEIKRTVAAPSV
underlined) FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQINKVDNALQSGNS
QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
CD79B, VTKSFNRGECHHHHHH
Polatuzumab,
B025-1_LC
CD79B, EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYVVIEVVVRQAPGK
62
Polatuzumab, GLEVVIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRA
B025-2_HC EDTAVYYCTRRVPIRLDYVVGQGTLVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHH
HHHH
Constructs for binding CD37
E200+CD37 GHNYTTRNILPGLNITSEIVLTQSPATLSLSPGERATLSCRASENV 63
binder (nfP2X7 YSYLAVVYQQKPGQAPRLLIYFAKTLAEGIPARFSGSGSGTDFTLTI
epitope SSLEPEDFAVYYCQHHSDNPVVTFGQGTKVEIKRTVAAPSVFIFPP
underlined) SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
C037, FNRGECHHHHHH
Otlertuzumab,
B026-1_LC
CD37, EVQLVQSGAEVKKPGESLKISCKGSGYSFTGYNMNVVVRQMPGK
64
Otlertuzumab, GLEVVMGN ID PYYGGTTYN RKFKGQVTISADKSISTAYLQWSSLKA
B026-2_HC SDTAMYYCARSVGPFDSVVGQGTLVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHH
H H HH
Constructs for binding CD38
E200+CD38 GHNYTTRNILPGLNITSEIVLTQSPATLSLSPGERATLSCRASQSV 65
binder (nfP2X7 SSYLAVVYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLT
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
epitope ISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFP
underlined) PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
CD38,
SFNRGECHHHHHH
Daratumumab,
B027-1_LC
CD38, EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGK
66
Daratumumab, GLEVVVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLR
B027-2_HC AEDTAVYFCAKDKILVVFGEPVFDYVVGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCHHHHHH
Constructs for binding CD70
E200+CD70 GHNYTTRNILPGLNITSQAVVTQEPSLTVSPGGTVTLTCGLKSGS 67
binder (nfP2X7 VTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNK
epitope AALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLKRTVAAPS
underlined) VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
CD70, PVTKSFNRGECHHHHHH
Cusatuzumab,
B028-1_LC
CD70, EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNVVVRQAPGK
68
Cusatuzumab, GLEVVVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLR
B028-2_HC AEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCHHHHHH
Constructs for binding CD30
E200+CD30 GHNYTTRNILPGLNITSDIVLTQSPASLAVSLGQRATISCKASQSV 69
binder (nfP2X7 DFDGDSYMNVVYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGT
epitope DFTLNIHPVEEEDAATYYCQQSNEDPVVTFGGGTKLEIKRTVAAPS
underlined) VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
CD30, PVTKSFNRGECHHHHHH
Brentuximab,
B029-1_LC
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
31
CD30, QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITVVVKQKPGQGL
70
Brentuximab, EWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSED
B029-2_HC TAVYFCANYGNYVVFAYWGQGTQVTVSAASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHH
HHHH
Constructs for binding C033
E200+0033 GHNYTTRNILPGLNITSDIQLTQSPSTLSASVGDRVTITCRASESLD 71
binder (nfP2X7 NYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGT
epitope EFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVAAPS
underlined) VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
CD33,
PVTKSFNRGECHHHHHH
Gemtuzumab,
B030-1_LC
C033, EVOLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHVVVRQAPGQS
72
Gemtuzumab, LEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSE
B030-2_Heavy DTAFYYCVNGNPVVLAYVVGQGTLVTVSSASTKGPSVFPLAPSSKS
Chain TSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHHH
HHH
E200+CD33 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASESV 73
binder (nfP2X7 DNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGT
epitope DFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIKRTVAAPS
underlined) VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
CD33,
PVTKSFNRGECHHHHHH
Lintuzumab,
B031-1_LC
CD33, QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHVVVRQAPG
74
Lintuzumab, QGLEVVIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLR
B031-2_HC SEDTAVYYCARGRPAMDYWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVN H KPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding Her2
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
32
E200-'-Her2 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCKASQDV
75
binder (nfP2X7 SIGVAVVYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLT
epitope ISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPS
underlined) DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
Her2, NRGECHHHHHH
Pertuzumab,
B032-1_LC
Her2, EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDVVVRQAPGK
76
Pertuzumab, GLEVVVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLR
B032-2_HC AEDTAVYYCARNLGPSFYFDYVVGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
E200+Her2 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASQDV
77
binder (nfP2X7 NTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLT
epitope ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP
underlined) SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
Her2,
FNRGECHHHHHH
Trastuzumab,
B033-1_LC
Her2, EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVVVRQAPGKG
78
Trastuzumab, LEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAE
B033-2_HC DTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
Constructs for binding EGFR
E200+EGFR GHNYTTRNILPGLNITSEIVMTQSPATLSLSPGERATLSCRASQSV 79
binder (nfP2X7 SSYLAVVYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLT
epitope ISSLEPEDFAVYYCHQYGSTPLTFGGGTKAEIKRTVAAPSVFIFPP
underlined) SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVIKS
EGFR, FNRGECHHHHHH
Necitumumab,
B034-1_LC
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
33
EGFR, QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPG
80
Necitumumab, KGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTA
B034-2_HC ADTAVYYCARVSIFGVGTFDYVVGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
E200+EGFR GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCSASSSV 81
binder (nfP2X7 TYMYVVYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTI
epitope SSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVAAPSVFIEPPS
underlined) DEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNSQESVT
EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
EGFR, NRGECHHHHHH
Matuzumab,
B035-1_LC
EGFR, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHVVVRQAPG
82
Matuzumab, QGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSL
B035-2_HC RSEDTAVYYCASRDYDYDGRYFDYVVGQGTLVTVSSASTKGPSV
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCHHHHHH
E200+EGFR GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCQASQDI 83
binder (nfP2X7 SNYLNVVYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTF
epitope TISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPP
underlined) SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
EGFR, FNRGECHHHHHH
Panitumumab,
B036-1_LC
EGFR, QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYVVTVVIRQSP
84
Pa nitumumab, GKGLEWIGH IYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAA
B036-2_HC DTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHH
HHHH
Constructs for binding CD276
E200+CD276 GHNYTTRNILPGLNITSEIVMTQSPATLSVSPGERVILSCRASQS1 85
binder (nfP2X7 SDYLYVVYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTI
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
34
epitope NSVEPEDVGVYYCQNGHSFPLTFGQGTKLELKRTVAAPSVFIFPP
underlined) SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
CO276, hu8H9-
FNRGECHHHHHH
6m, B037-1_LC
0D276, hu8H9- QVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINVVVRQRPGQ
86
6m, B037-2_HC GLEVVIGWIFPGDDSTQYNEKFKGKATLTTDTSTSTAYMELSSLRS
EDTAVYFCARQTTGTVVFA'YVVGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding GD2
E200+GD2 binder GHNYTTRNILPGLNITSKIVMTQTPATLSVSAGERVTITCKASQSV
87
(nfP2X7 epitope SNHVTVVYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTF
underlined) TISSVQSEDFAVYFCQQDYSSFGQGTKLEIKRTVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
GD2, Naxitamab' QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN
B038-1_LC RGECHHHHHH
GD2, Naxitamab, QVQLVESGPGVVQPGRSLRLSCAVSGFSVTNYGVHWVRQPPGK
88
B038-2_HC GLEVVLGVIWAGGITNYNSSVKGRLTISKDNSKNTVYLQMNSLRAE
DTAVYYCASRGGHYGYALDYVVGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding BCMA
E200+BCMA GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCSASQDI 89
binder (nfP2X7 SNYLNVVYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTL
epitope TISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTVAAPSVFIFP
underlined) PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
BCMA, clone CA8 SFNRGECHHHHHH
J9M0, B039-1_LC
BCMA, clone CA8 QVQLVQSGAEVKKPGSSVKVSCKGSGYTFTNYWMHVVVRQAPG
90
J9M0, B039- QGLEWIGATYRGHSDTYYNQKFKGRATLTADTSTSTAYMELSSL
2_HC RSEDTAVYYCTRGAIYDGYDVLDNWGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCHHHHHH
Constructs for binding CD371
E200+CD371 GHNYTTRNILPGLNITSDIVMTOSPSSVSASVGDRVTITCRASC)DI 91
binder (nfP2X7 SSVVLAWYQQKPGKAPKLLIYAASSLQSGVPSRFNGSGSGTDFTL
epitope TISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFP
underlined) PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
US10568947 CA
¨ SFNRGECHHHHHH
R9, B040-1_LC
CD371, QVQLVQSGAEVKEPGASVKVSCKAPANTFSDHVMHVVVRQAPG
92
QRFEWMGYIHAANGGTHYSQKFQDRVTITRDTSANTVYMDLSSL
US10568947 CA
¨ RSEDTAVYYCARGGYNSDAFDIWGQGTMVTVSSASTKGPSVFPL
R9, B040-2_HC
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCHHHHHH
Constructs for binding CD135
E200+CD135 GHNYTTRNILPGLNITSDIVLTQSPATLSVTPGDSVSLSCRASQSIS 93
binder (nfP2X7 NNLHVVYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSIN
epitope SVETEDFGVYFCQQSNTWPYTFGGGTKLEIKRTVAAPSVFIFPPS
underlined) DEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNSQESVT
EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
CD135, clone NRGECHHHHHH
4G8, B041-1_LC
CD135, clone
QVQLQQPGAELVKPGASLKLSCKSSGYTFTSYWMHVVVRQRPGH 94
4G8, B041-2_HC GLEVVIGEIDPSDSYKDYNQKFKDKATLTVDRSSNTAYMHLSSLTS
DDSAVYYCARAITTTPFDFWGQGTTLTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCH
HHHHH
Constructs for binding CD123
E200+CD123 GHNYTTRNILPGLNITSDIVLTOSPASLAVSLGQRATISCRASESVD 95
binder (nfP2X7 NYGNTFMHVVYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTD
epitope FTLTINPVEADDVATYYCQQSNEDPPTFGAGTKLELKRTVAAPSV
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
36
underlined) FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNS
QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
C0123, clone
VTKSFNRGECHHHHHH
32716, B042-
1 LC
CD123, clone
QIQLVQSGPELKKPGETVKISCKASGYIFTNYGMNVVVKQAPGKSF 96
32716, B042- KWMGWINTYTGESTYSADFKGRFAFSLETSASTAYLHINDLKNED
2_HC TATYFCARSGGYDPMDYWGQGTSVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCH
HHHHH
Constructs for binding CD105
E200+C D 105 GHNYTTRN ILPGLN
ITSQIVLSQSPAILSASPGEKVTMTCRASSSV 97
binder (nfP2X7 SYMHVVYQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSL
epitope TISRVEAEDAATYYCQQWSSNPLTFGAGTKLELKRTVAAPSVFIF
underlined) PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
0D105, KSFNRGECHHHHHH
Carotuximab,
B043-1_LC
CD105, EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAVVMDVVVRQSPE 98
Carotuximab, KGLEWVAEIRSKASNHATYYAESVKGRFTISRDDSKSSVYLQMNS
B043-2_HC LRAEDTGIYYCTRWRRFFDSWGQGTTLTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding ROR-1
E200+ROR-1 GHNYTTRNILPGLNITSEIVLSQSPAITAASLGQKVTITCSASSNVS 99
binder (nfP2X7 YIHWYQQRSGTSPRPWIYEISKLASGVPVRFSGSGSGTSYSLTIS
epitope SMEAEDAAIYYCQQWNYPLITFGSGTKLEIQRTVAAPSVFIFPPSD
underlined) ROR- EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
1, clone Dl 0v3, QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN
B044-1_LC RGECHHHHHH
ROR-1, clone
QVQLKESGPGLVAPSQTLSITCTVSGFSLTSYGVHVVVRQPPGKG 100
D10v3, B044- LEWLGVIWAGGFTNYNSALKSRLSISKDNSKSQVLLKMTSLQTDD
2_HC TAMYYCARRGSSYSMDYVVGQGTSVTVSSASTKGPSVFPLAPSS
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
37
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCH
HHHHH
Constructs for binding PD-L1
E200+PD-L1 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASQDV 101
binder (nfP2X7 STAVAVVYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT
epitope ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPP
underlined) PD- SDEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNSQESV
Li, Atezolizumab, TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
B045-1_LC FNRGECHHHHHH
PD-L1, EVQLVESGGGLVQPGGSLRLSCAASGFTESDSVVIHVVVRQAPGK
102
Atezolizumab, GLEVVVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLR
B045-2_HC AEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
Constructs for binding MET-R
E200+MET-R GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCKSSQSL 103
binder (nfP2X7 LYTSSQKNYLAVVYQQKPGKAPKLLIYVVASTRESGVPSRFSGSGS
epitope GTDFTLTISSLQPEDFATYYCQQYYAYPVVTFGQGTKVEIKRTVAA
underlined) MET- PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
R, Onartuzumab, GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
B046-1_LC LSSPVTKSFNRGECHHHHHH
MET-R, EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYVVLHWVRQAPGK
104
Onartuzumab, GLEVVVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLR
B046-2_HC AEDTAVYYCATYRSYVTPLDYVVGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
Constructs for binding PDGFRalpha
E200+PDGFR GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVSITCRPSQSF 105
binder (nfP2X7 SRYINWYQQKPGKAPKLLIHAASSLVGGVPSRFSGSGSGTDFTLT
epitope ISSLQPEDFATYYCQQTYSNPPITFGQGTRLEMKRTVAAPSVFIFP
underlined) PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
PDGFRalpha, VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
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Tovetumab, SFNRGECHHHHHH
B047-1_LC
PDGFRalpha, QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMNWIRQAPGK 106
Tovetumab, GLEVVVSYISSSGSI IYYADSVKGRFTISRDNAKNSLYLQMNSLRAE
B047-2_HC DTAVYYCAREGRIAARGMDVWGQGTTVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
E200+PDGFR GHNYTTRNILPGLNITSEIVLTQSPATLSLSPGERATLSCRASQSV 107
binder (nfP2X7 SSYLAVVYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLT
epitope ISSLEPEDFAVYYCQQRSNWPPAFGQGTKVEIKRTVAAPSVFIFP
underlined) PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
PDGFRalpha, VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
Olaratumab, SFNRGECHHHHHH
B048-1_LC
PDGFRalpha, QLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYVVGWLRQSPG 108
Olaratumab, KGLEWIGSFFYTGSTYYNPSLRSRLTISVDTSKNQFSLMLSSVTAA
B048-2_HC DTAVYYCARQSTYYYGSGNYYGWFDRWDQGTLVTVSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
KKVEPKSCHHHHHH
Constructs for binding Her3
E200+Her3 GHNYTTRNILPGLNITSQSALTQPASVSGSPGQSITISCTGTSSDV
109
binder (nfP2X7 GSYNVVSWYQQHPGKAPKLIIYEVSQRPSGVSNRFSGSKSGNTA
epitope SLTISGLQTEDEADYYCCSYAGSSIFVIFGGGTKVTVLRTVAAPSV
underlined) Her3, FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS
Seribantumab, QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
B049-1_LC VTKSFNRGECHHHHHH
Her3, EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMAWVRQAPGK
110
Seribantumab, GLEWVSSISSSGGWTLYADSVKGRFTISRDNSKNTLYLQMNSLR
B049-2_HC AEDTAVYYCTRGLKMATIFDYVVGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding FRalpha
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E200+FRa binder GHNYTTRNILPGLNITSDIQLTQSPSSLSASVGDRVTITCSVSSSIS
111
(nfP2X7 epitope SNNLHVVYQQKPGKAPKPVVIYGTSNLASGVPSRFSGSGSGTDYT
underlined) FTISSLQPEDIATYYCQQWSSYPYMYTFGQGTKVEIKRTVAAPSV
FRalpha, FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNS
Farletuzumab, QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
B050-1_LC VTKSFNRGECHHHHHH
FRalpha, EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSVVVRQAPGK
112
Farletuzumab, GLEVVVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLR
B050-2_HC PEDTGVYFCARHGDOPAVVFAYVVGQGTPVTVSSASTKGPSVFPL
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCHHHHHH
Constructs for binding GPC3
E200+GPC3 GHNYTTRNILPGLNITSDVVMTQSPLSLPVTPGEPASISCRSSQSL 113
binder (nfP2X7 VHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSG
epitope TDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRTVAAP
underlined) SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
GPC3, NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
Codrituzumab, SSPVTKSFNRGECHHHHHH
B051-1_LC
GPC3, QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHVVVRQAPGQ
114
Codrituzumab, GLEVVMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLT
B051-2_HC SEDTAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHH
HHHH
Constructs for binding SLAMF7
E200+SLAMF7 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCKASQDV 115
binder (nfP2X7 GIAVAVVYQQKPGKVPKLLIYVVASTRHTGVPDRFSGSGSGTDFTL
epitope TISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIKRTVAAPSVFIFP
underlined) PSDEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNSQES
SLAMF7, VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
Elotuzumab, SFNRGECHHHHHH
B052-1_LC
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SLAMF7, EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYVVMSVVVRQAPG
116
Elotuzumab, KG LEWIGE IN PDSSTINYAPSLKDKFI ISRDNAKNSLYLQMNSLRAE
B052-2_HC DTAVYYCARPDGNYWYFDVWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding TNFRSF1OB
E200+ GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCKASQDV
117
INFRSF10B GTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTL
binder (nfP2X7 TISSLQPEDFATYYCQQYSSYRTFGQGTKVEIKRTVAAPSVFIFPP
epitope SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
underlined), TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
Tigatuzumab, FNRGECHHHHHH
B053-1_LC
TN FRSF10B, EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYVMSVVVRQAPGK
118
Tigatuzumab, GLEVVVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMNSLRA
B053-2_HC EDTAVYYCARRGDSMITTDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding GPNMB
E200+ GPNMB GHNYTTRNILPGLNITSEIVMTQSPATLSVSPGERATLSCRASQSV
119
binder (nfP2X7 DNNLVVVYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLT
epitope ISSLQSEDFAVYYCQQYNNWPPWTFGQGTKVEIKRTVAAPSVFIF
underlined), PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
GPN MB, SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
Glembatumumab, KSFNRGECHHHHHH
B054-1_LC
GPN MB, QVQLQESGPGLVKPSQTLSLTCTVSGGSISSFNYYWSWIRHHPG
120
Glembatumumab, KG LEWIGYIYYSGSTYSN PSLKSRVTISVDTSKNQFSLTLSSVTAA
B054-2_HC DTAVYYCARGYNWNYFDYWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVN H KPSNTKVDKKVEPKSC
HHHHHH
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Constructs for binding VEGFR2
E2004 VEGFR2 GHNYTTRN ILPGLN ITSD I Q MTQSPSSVSASIGDRVTITCRASQGID 121
binder (nfP2X7 NWLGWYQQKPGKAPKLLIYDASNLDTGVPSRFSGSGSGTYFTLTI
epitope SSLQAEDFAVYFCQQAKAFPPTFGGGTKVD I KRTVAAPSVFI FPP
underlined), SD EQ LKSGTASVVC LLN NFYPREAKVQVVKVDNALQSGNSQESV
Ra mu ciru ma b, TEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTH QGLSSPVTKS
B055-1_LC FNRGECHHHHHH
VEGFR2, EVQLVQSGGG LVKPGGSLRLSCAASGFTFSSYSM NVVVRQAPGK
122
Ramucirumab, GLEVVVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRA
B055-2_HC EDTAVYYCARVTDAFDIVVGQGTMVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCHH
H H HH
Constructs for binding a467 Wor aE[37
E200+ a4137 and GHNYTTRN ILPGLN ITSD I Q MTQSPSSLSASVGDRVTITCRASESV
123
aE137binder DD LLHVVYQQKPGKAPKLLIKYASQ SI SGVPSRFSG SGSGTDFTLT
(nfP2X7 epitope ISSLQPEDFATYYCQQGNSLPNTFGQGTKVEIKRTVAAPSVFIFPP
underlined), SD EQ LKSGTASVVC LLN NFYPREAKVQWKVDNALQSGNSQESV
Etrol izu ma b, TEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTH QGLSSPVTKS
B056-1_LC FNRGECHHHHHH
a437 & 0E137,
EVQLVESGGGLVQPGGSLRLSCAASG FFITNNYVVGVVVRQAPGK 124
Etrolizumab, GLEVVVGYISYSGSTSYN PSLKSRFTISRDTSKNTFYLQMNSLRAE
B056-2_HC DTAVYYCARTGSSGYFD FWGQGTLVTVSSASTKG PSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SG LYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCH
HHHHH
E200+ a4137
GHNYTTRN ILPGLN ITSD IQ MTQSPSSVSASVGDRVTITCRASQG I 125
binder (nfP2X7 SSVVLAWYQQKPGKAPKLLIYGASN LESGVPSRFSGSGSGTDFTL
epitope TI SSLQ PED FANYYCQQAN SFPVVTFGQGTKVEI KRTVAAPSVF I
F
underlined), a4 [37 , PPSD EQLKSGTASVVCLLN N FYPREAKVQWKVDNALQSG NSQE
Abrilumab, B057- SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
1_LC KSFNRGECHHHHHH
a4137, Abrilumab, QVQLVQSGAEVKKPGASVKVSCKVSGYTLSDLSIHWVRQAPG KG
126
LEWMGGFDPQDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLKS
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B057-2_HC EDTAVYYCATGSSSSWFDPWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding CSPG4
E200+ CSPG4 GHNYTTRNILPGLNITSRSTQSALTQPASVSGSPGQSITISCTGTS
127
binder (nfP2X7 SDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSKRFSGSKS
epitope GNTASLTISGLQAEDEADYYCSSYTSSSTRHVFGTGTQLTVLGRT
underlined), VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
CSPG4, D2A- QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
1h10-UC12, QGLSSPVTKSFNRGECHHHHHH
B058-1_LC
CSPG4, D2A- EVQLVESGAEVKKPGDSLKISCKGSGYSFTSYWIGVVVRQMPGK
128
1h10-UC12, GLEVVMGIIYPGDSVTTYSPAFQGDVTISVDKSISTAYLQWNSLKA
B058-2_HC SDTGIYYCARRRGNYYMDVVVGNGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSINNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding CD80
E200+ CD80 GHNYTTRNILPGLNITSESALTQPPSVSGAPGQKVTISCTGSTSNI
129
binder (nfP2X7 GGYDLHVVYQQLPGTAPKLLIYDINKRPSGISDRFSGSKSGTAASL
epitope Al TGLQTEDEADYYCQSYDSSLNAQVFGGGTRLTVLRTVAAPSVF
underlined), IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
Galiximab, B059- ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
1_LC TKSFNRGECHHHHHH
CD80, Galiximab, QVQLQESGPGLVKPSETLSLTCAVSGGSISGGYGWGWIRQPPG
130
B059-2_HC KGLEWIGSFYSSSGNTYYNPSLKSQVTISTDTSKNQFSLKLNSMT
AADTAVYYCVRDRLFSVVGMVYNNVVFDVWGPGVLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCHHHHHH
Constructs for binding CCR4
E200+ CCR4 GHNYTTRNILPGLNITSDVLMTQSPLSLPVTPGEPASISCRSSRNI
131
binder (nfP2X7 VHINGDTYLEVVYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSG
epitope TDFTLKISRVEAEDVGVYYCFQGSLLPWTFGQGTKVEIKRTVAAP
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underlined), SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
Mogamulizu ma b, NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
B060-1_LC SSPVTKSFNRGECHHHHHH
CCR4, EVQLVESGGDLVQPGRSLRLSCAASGFIFSNYGMSVVVRQAPGK
132
Mogamulizu ma b, GLEVVVATISSASTYSYYPDSVKGRFTISRDNAKNSLYLQMNSLRV
B060-2_HC EDTALYYCGRHSDGNFAFGYVVGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding CD115
E200+ C D115 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASEDV
133
binder (nfP2X7 NTYVSVVYQQKPGKAPKLLIYAASNRYTGVPSRFSGSGSGTDFTL
epitope TISSLQPEDFATYYCQQSFSYPTFGQGTKLEIKRTVAAPSVFIFPP
underlined), SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
CD115-CSF-1R, TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
Emactuzumab, FNRGECHHHHHH
B061-1_LC
CD115-CSF-1R, QVQLVQSGAEVKKPGASVKVSC KASGYTFTSYD I SVVVRQAPGQ
134
Emactuzumab, GLEWMGVIVVTDGGTNYAQKLQGRVTMTTDTSTSTAYMELRSLR
B061-2_HC SDDTAVYYCARDQRLYFDVVVGQGTTVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSINNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVN H KPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding ENOX-2
E200+ ENOX-2 GHNYTTRNILPGLNITSENVLTQSPAIMSASPGERVTMTCSASSSI
135
binder (nfP2X7 RYIYVVYQQKPGSSPRLLIYDTSNVAPGVPFRFSGSGSGTSYSLTI
epitope NRMEAEDAATYYCQEWSGYPYTEGGGTKLELKRTVAAPSVFIFP
underlined), PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
ENOX-2, VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
US9459256, SFNRGECHHHHHH
B062-1_LC
ENOX-2, EVKLQESGTEVVKPGASVKLSCKASGYIFTSYDIDVVVRQTPEQGL
136
US9459256, EVVIGWIFPGEGSTEYNEKFKGRATLSVDKSSSTAYMELTRLTSED
B062-2_HC SAVYFCARGDYYRRYFDLWGQGTTVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCH
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HHHHH
Constructs for binding CD56
E200+ 0D56 GHNYTTRNILPGLNITSDVVMTQSPLSLPVTLGQPASISCRSSQIII
137
binder (nfP2X7 HSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSG
epitope TDFTLKISRVEAEDVGVYYCFQGSHVPHTFGQGTKVEIKRTVAAP
underlined), SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
CD56, NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
Lorvotuzumab, SSPVTKSFNRGECHHHHHH
B063-1_LC
CD56, QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGK
138
Lorvotuzumab, .. GLEVVVAYISSGSFTIYYADSVKGRFTISRDNSKNTLYLQMNSLRA
B063-2_HC EDTAVYYCARMRKGYAMDYVVGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding huVH1-69
E200+ huVH1-69 GHNYTTRNILPGLNITSDIQLTQSPSSLSASVGDRVTITCRASQGIS
139
binder (nfP2X7 SNIVWLQQKPGKAPKGLIYHGTNLESGVPSRFSGSGSGTDYTLTI
epitope SSLEPEDFATYYCVQYSQFPPTFGQGTKLEIKRTVAAPSVFIFPPS
underlined), DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
huVH1-69, B075- EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
1_LC NRGECHHHHHH
huVH1-69, B075- QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYVVMHVVVKQAPG
140
2_HC QGLEWIGAVSPGNSDTSYNEKFKGKATLTVDTSASTAYMELSSL
RSEDTAVYYCTRSRYGNNALDYVVGQGTLVTVSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
Constructs for binding CD19 (IgG1 format)
CD19, EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHVVVRQAPGK
141
Tafasitamab, GLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSE
DTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLA
0R19_1, wt-IgG1,
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
HC
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
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VDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEINESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK
SLSLSPGKHHHHHH
CD19, EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHVVVRQAPGK
142
Tafasitamab, GLEVVIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSE
DTAMYYCARGTYYYGTRVFDYVVGQGTLVTVSSASTKGPSVFPLA
OR19_2, IgG1-
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
SD 1E, HC
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEVVESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALH N HYTQ
KSLSLSPGKHHHH HI-I
Constructs for binding CD19 (Fab format)
CD19, EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHVVVRQAPGK
143
Tafasitamab, GLEVVIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSE
B020-2_Heavy DTAMYYCARGTYYYGTRVFDYVVGQGTLVTVSSASTKGPSVFPLA
chain PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SC HHH HHH
CD19, DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQK
144
Tafasitamab, PGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFA
VYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
OR19_7 SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Light chain
E200+ CD19 GHNYTTRNILPGLNITSDIVMTQSPATLSLSPGERATLSCRSSKSL
145
binder (nfP2X7 QNVNGNTYLYINFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGS
epitope GTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPS
underlined), VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
CD19, SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
Tafasitamab, PVTKSFNRGEC
Light chain
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
46
OR19_8
E200+ CD19 GHNYTTRNILPGLNITSGGGGSDIVMTQSPATLSLSPGERATLSC
146
binder (nfP2X7 RSSKSLQNVNGNTYLYVVFQQKPGQSPQLLIYRMSNLNSGVPDRF
epitope SGSGSGTEFTLTISSLEPEDFAVYYC MOH LEYPITFGAGTKLEIKR
underlined), TVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQVVKVDNA
CD19, LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
Tafasitamab, HQGLSSPVTKSFNRGEC
Light chain
OR19_9
E200+ CD19 GHNYTTRNILPGLNITSGGGGSGGGGSGGGGSDIVMTQSPATLS
147
binder (nfP2X7 LSPGERATLSCRSSKSLQNVNGNTYLYVVFQQKPGQSPQLLIYRM
epitope SNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPI
underlined), TFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR
CD19, EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY
Tafasitamab, EKHKVYACEVTHQGLSSPVTKSFNRGEC
Light chain
0R19_10
E200+ CD19 GHNYTTRNILPGLNITSTFHKTSGSGKDIVMTQSPATLSLSPGERA
148
binder (nfP2X7 TLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGV
epitope PDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKL
underlined), El KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
CD19, VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
Tafasitamab, CEVTHQGLSSPVTKSFNRGEC
Light chain
OR19_11
E200+ CD19 GHNYTTRNILPGLNITSTFHKTDIVMTQSPATLSLSPGERATLSCR
149
binder (nfP2X7 SSKSLQNVNGNTYLYVVFQQKPGQSPQLLIYRMSN LNSGVPDRFS
epitope GSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRT
underlined), VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
CD19, QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
tafasitamab, Light QGLSSPVTKSFNRGEC
chain
OR19_12
E200+ CD19 GHNYTTRNILPGLNITSTFHKTGSDIVMTQSPATLSLSPGERATLS
150
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
47
binder (nfP2X7 CRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPD
epitope RFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEI
underlined), KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
CD19, NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
Tafasitamab, VTHQGLSSPVTKSFNRGEC
Light chain
OR19_13
E200+ 0019 GHNYTTRNILPGLNITSTFHGSDIVMTQSPATLSLSPGERATLSCR
151
binder (nfP2X7 SSKSLONVNGNTYLYWFQQKPGQSPQLLIYRMSN LNSGVPDRFS
epitope GSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRT
underlined), VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
CD19, QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
Tafasitamab, QGLSSPVTKSFNRGEC
Light chain
OR19_14
E200+ 0019 GHNYTTRNILPGLNITSGSDIVMTQSPATLSLSPGERATLSCRSSK
152
binder (nfP2X7 SLQNVNGNTYLYVVFQQKPGQSPQLLIYRMSNLNSGVPDRFSGS
epitope GSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVA
underlined), APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
CD19, GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
Tafasitamab, LSSPVTKSFNRGEC
Light chain
OR19_15
E200+ 0019 DFPGHNYTTRNILPGLNITSGSDIVMTQSPATLSLSPGERATLSCR
153
binder (nfP2X7 SSKSLQNVNGNTYLYVVEQQKPGQSPQLLIYRMSNLNSGVPDRPS
epitope GSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRT
underlined), VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
0019, QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
Tafasitamab, QGLSSPVTKSFNRGEC
Light chain
OR19_NEW_001
E200+ 0019 DFPGHNYTTRNILPGLNITSGGGGSDIVMTQSPATLSLSPGERATL
154
binder (nfP2X7 SCRSSKSLONVNGNTYLYWFQQKPGQSPOLLIYRMSNLNSGVP
epitope DRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLE
underlined), IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
CD19, DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
Tafasitamab, EVTHQGLSSPVTKSFNRGEC
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
48
Light chain
OR19_NEW_002
E200+ CD19 DFPGHNYTTRNILPGLNITSGGGGSGGGGSDIVMTQSPATLSLSP
155
binder (nfP2X7 GERATLSCRSSKSLQNVNGNTYLYVVFQQKPGQSPOLLIYRMSNL
epitope NSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFG
underlined), AGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
CD19, VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
Tafasitamab, KVYACEVTHQGLSSPVTKSFNRGEC
Light chain
OR19_NEW_003
E200+ CD19 DFPGHNYTTRNILPGLNITSGGGGSGGGGSGGGGSDIVMTQSPA
156
binder (nfP2X7 TLSLSPG ERATLSCRSSKSLQNVNGNTYLYVVFQQKPGQSPQLLI
epitope YRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHL
underlined), EYPITFGAGTKLEIKRTVAAPSVF IF PPSD EQLKSGTASVVCLLN N
F
CD19, YPREAKVQVVKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
Tafasitamab, ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Light chain
OR19_NEW_004
E200+ CD19 DFPGHNYTTRNILPGLNITSTFHKTSGSGKDIVMTQSPATLSLSPG
157
binder (nfP2X7 ERATLSCRSSKSLQNVNGNTYLYVVFQQKPGQSPQLLIYRMSNLN
epitope SGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGA
underlined), GTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
CD19, QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK
Tafasitamab, VYACEVTHQGLSSPVTKSFNRGEC
Light chain
OR19_NEW_005
E200+ CD19 DFPGHNYTTRNILPGLNITSTFHKTSGSGKGSDIVMTQSPATLSLS
158
binder (nfP2X7 PG ERATLSCRSSKSLQNVNGNTYLYVVFQQKPGQSPOLLIYRMSN
epitope LNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITF
underlined), GAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EA
CD19, KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
Tafasitamab, HKVYACEVTHQGLSSPVTKSFNRGEC
Light
chain0R19_NEW
006
_
E200+ CD19 DFPGHNYTTRNILPGLNITSTFHKTSGSGKGGGGSDIVMTQSPAT
159
binder (nfP2X7 LSLSPGERATLSCRSSKSLQNVNGNTYLYVVFQQKPGQSPQLLIY
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
49
epitope RMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLE
underlined), YPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY
CD19, PREAKVQVVKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
Tafasitamab, DYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Light chain
OR19_NEW_007
E200+ CD19 DFPGHNYTTRNILPGLNITSTFHGGGGSDIVMTQSPATLSLSPGE
160
binder (nfP2X7 RATLSCRSSKSLQNVNGNTYLYVVFQQKPGQSPQLLIYRMSNLNS
epitope GVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAG
underlined), TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
0019, WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
Tafasitamab, YACEVTHQGLSSPVTKSFNRGEC
Light chain
OR19_NEW_008
E200+ CD19 GHNYTTRNILPGLNITSTFHGGGGSDIVMTQSPATLSLSPGERATL
161
binder (nfP2X7 SCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVP
epitope DRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLE
underlined), IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
CD19, DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
Tafasitamab, EVTHQGLSSPVTKSFNRGEC
Light chain
OR19_NEW_009
E200+ CD19 DFPGHNYTTRNILPGLNITSTFHKTGGGGSDIVMTQSPATLSLSPG
162
binder (nfP2X7 ERATLSCRSSKSLQNVNGNTYLYVVFQQKPGQ SPOLLIYRMSN LN
epitope SGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQH LEYPITFGA
underlined), GTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
CD19, QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK
Tafasitamab, VYACEVTHQGLSSPVTKSFNRGEC
Light chain
OR19_NEW_010
E200+ CD19 GHNYTTRN ILPGLN ITSTFH KTGGGGSDIVMTQSPATLSLSPG ER
163
binder (nfP2X7 ATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSG
epitope VPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQH LEYPITFGAGT
underlined), KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
CD19, WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
Tafasitamab, YACEVTHQG LSSPVTKSFNRGEC
Light chain
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
OR19_NEW_011
E200+ CD19 GHNYTTRN ILPGLN ITSGGGGSGGGGSDIVMTQSPATLSLSPG ER
164
binder (nfP2X7 ATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSG
epitope VPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQH LEYPITFGAGT
underlined), KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
CD19, WKVDNALQSG NSQESVTEQ DSKDSTYSLSSTLTLSI<A DYEKH KV
Tafasitamab, YACEVTHQG LSSPVTKSFN RG EC
Light chain
OR19_NEW_012
Exemplary CAR constructs
CAR7 (anti- MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAA
165
nfP2X7) SG FTFR N H DMGWVRQAPGKGLEWVSAISGSGGSTYYANSVKGR
FTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTEFDYRSP
GTLVTVSSRAAAI EVMYPPPYLD NEKSNGTI I HVKG KH LC PSPLFP
GPSKPFVVVLVVVGGVLACYSLLVTVAFIIFVVVRSKRSRLLHSDYM
NMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFM
RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQ
NQLYN ELN LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN
ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL
HMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCE
LPHPAFLLIPRKVCNGIG I GEFKDSLSINATNIKH FKNCTSISGD LHI
LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDL
HAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVII SG
NKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALC
SPEGCWG PEPRDCVSC RN VS RGREC VD KC N LLEG EPREFVENS
EC IQCHPECLPQAMNITCTGRGPD NCIQCAHYIDGPHCVKTCPAG
VMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGP
KIPSIATGMVGALLLLLVVALGIGLFM
CAR10 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAA
166
SG FTFRNH DMGWVRQAPGKGLEVVVSAISGSGGSTYYANSVKGR
(anti-nfP2X7) FTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTEFDYRSP
GTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT
RGLDFACDFVVVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHS
DYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQ
PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYK
QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE
GLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD
TYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTS
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
51
LLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSIS
GDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPEN
RTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGD
VI ISGNKNLCYANTI NWKKLFGTSGQKTKIISNRGENSCKATGQVC
HALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREF
VENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKT
CPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGC
PTNGPKIPSIATGMVGALLLLLVVALGIGLFM
CAR16 MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAA
167
SGFTFRNHDMGWVRQAPGKGLEWVSAISGSGGSTYYANSVKGR
(anti-nfP2X7) FTISRDNSKNTLYLQMNSLRAEDTAVYYCAEPKPMDTEFDYRSP
GTLVTVSSESKYGPPCPPCPFVVVLVVVGGVLACYSLLVTVAFIIF
VVVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV
KFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEM
GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG
LYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEEN
PGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINAT
NIKHFKNCTSISGDLH ILPVAFRGDSFTHTPPLDPQELD ILKTVKEIT
GFLLIQAWPEN RTDLHAFENLEIIRGRTKQHGQFSLAVVSLN ITSL
GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRG
ENSCKATGQVCHALCSPEGCVVGPEPRDCVSCRNVSRGRECVD
KCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQC
AHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCT
YGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM
Description Sequence
SEQ ID NO:
Constructs for binding to CD117
E200+CD117 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASQSI 169
binder (nfP2X7 NSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
epitope TISSLQPEDFATYYCQQGVSDITFGGGTKVEIKRTVAAPSVFIFPP
underlined), SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
W02019084067, TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
mAb-55, B076- FNRGECHHHHHH
1_LC
CD117, QVQLVQSGAEVKKPGSSVKVSCKASGGTFRIYAISWVRQAPGQG
170
W02019084067, LEWMGGIIPDFGVANYAQKFQGRVTITADESTSTAYMELSSLRSE
mAb-55, B076- DTAVYYCARGGLDTDEFDLWGRGTLVTVSSASTKGPSVFPLAPS
CA 03211323 2023- 9-7

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52
2_HC SKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding to C0133
E200+CD133 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASQGS 171
binder (nfP2X7 SYVAVVYQQKPGKAPKLLIYSASYLYSGVPSRFSGSRSGTDFTLTI
epitope SSLQPEDFATYYCQQGVWSLITFGQGTKVEIKRTVAAPSVFIFPPS
underlined), DEQLKSGTASVVCLLNNFYPREAKVQINKVDNALQSGNSQESVT
CA2962157, EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
RW03, B077- NRGECHHHHHH
1_LC
CD133, EVQLVESGGGLVQPGGSLRLSCAASGFNLSSSSIHVVVRQAPGK
172
CA2962157, GLEWVAYIYPYYSYTYYA DSVKG R FTISADTSKNTAYLQM NSLRA
RW03, B077- EDTAVYYCAREGSVAGEDYVVGQGTLVTVSSASTKGPSVFPLAPS
2_HC SKSTSGGTAALGCLVKDYFPEPVTVSINNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding to MUC1
E200+MUC1 GHNYTTRNILPGLNITSDIVMTQSPDSLAVSLGERATINCKSSQSL 173
binder (nfP2X7 LNSGDQKNYLTVVYQQKPGQPPKLLIYVVASTRESGVPDRFSGSG
epitope SGTDFTLTISSLQAEDVAVYYCQNDYSYPLTFGQGTKVEIKRTVA
underlined), APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQS
W02016130726A GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
1, B078-1_LC LSSPVTKSFNRGECHHHHHH
MUC1, QVQLVQSGAEVKKTGSSVKVSCKASGYTFTDHAIHVVVRQAPGQ
174
W02016130726A ALEWMGHFSPGNTDIKYNDKFKGRVTLTVDRSMSTAYMELSSLR
1, B078-2_HC SEDTAMYYCKTSTFFFDYVVGQGTMVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCH
HHHHH
Constructs for binding to Mesothelin (MSLN)
E200+MSLN GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASQSI 175
binder (nfP2X7 SSYLNWYQQKPGKAPKLLIYAASSLQSGVPSGFSGSGSGTDFTL
epitope TISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFP
underlined), PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
CA 03211323 2023- 9-7

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PCT/AU2022/050206
53
W02009120769, VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
B079-1_LC SFNRGECHHHHHH
MSLN, QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYY1NSWIRQPP
176
W02009120769, GKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTA
B079-2_HC ADTAVYYCAREGKNGAFD IVVGQGTMVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding to ROR2
E200+ROR2 GHNYTTRNILPGLNITSQSALTQPASVSGSPGQSITISCTGTSGDV 177
binder (nfP2X7 GGYNYVSVVYQHHPGKAPKLIIYDVNKRPSGFSDRFSGSKSGNTA
epitope SLTISGLQAEDEADYYCSSYTSTSTVFGGGTKLTVLGKRTVAAPS
underlined), VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
W02016142768A SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
1, B080-1_LC PVTKSFNRGECHHHHHH
ROR2, QITLKESGPELVKPTQTLTLTCTFSGFSLSTSGMSVSWIRQPPGK
178
W02016142768A ALEVVLARIDVVDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNTDPV
1, B080-2_HC DTATYYCARGFYLAYGSYDSWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding to IL13Ra2
E200+IL13Ra2 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCTASLSV
179
binder (nfP2X7 SSTYLHWYQQKPGSSPKLWIYSTSNLASGVPSRFSGSGSGTSFT
epitope LTISSLQPEDFATYYCHQYHRSPLTFGGGTKVEIKRTVAAPSVFIF
underlined), PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
W02014072888A SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
1, B081-1_LC KSFNRGECHHHHHH
IL13Ra2, EVQLVESGGGLVQPGGSLRLSCAASGFSLTKYGVHWVRQAPGK
180
W02014072888A GLEVVVGVKWAGGSTDYNSALMSRFTISKDNAKNSLYLQMNSLR
1, B081-2_HC AEDTAVYYCARDHRDAMDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
CA 03211323 2023- 9-7

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Constructs for binding to IL13Ra2
IL13Ra2, ligand, HHHHHHGPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLT
181
US20180265844, AGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHV
B082 RDTKIEVAQFVKDLLLHLKKLFREGRFNGGGGSGHNYTTRNILPG
LNITS
Constructs for binding to EPHA2
E200+EPHA2 GHNYTTRNILPGLNITSDIQLTQSPSSLSASVGDRVTITCKASQDIN 182
binder (nfP2X7 NYLSWYQQKPGQAPRLLIYRANRLVDGVPDRFSGSGYGTDFTLTI
epitope NNIESEDAAYYFCLKYDVFPYTFGQGTKVEIKRTVAAPSVFIFPPS
underlined), DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
W02007073499, EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
B083-1_LC NRGECHHHHHH
EPHA2, QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSVVVRQAPGQ
183
W02007073499, ALEWMGTISSGGTYTYYPDSVKGRFTISRDNAKNSLYLQMNSLR
B083-2_HC AEDTAVYYCAREAIFTYWGRGTLVTSSASTKGPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHHHH
HH
Constructs for binding to EGFRvIll
E200+EGFRvl II GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASQGI
184
binder (nfP2X7 RNNLAVVYQQKPGKAPKRLIYAASNLQSGVPSRFTGSGSGTEFTLI
epitope VSSLQPEDFATYYCLQHHSYPLTSGGGTKVEIKRTVAAPSVFIFPP
underlined), SDEQLKSGTASVVOLLNNIFYPREAKVQVVKVDNALQSGNSQESV
W02013185010, TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
B084-1_LC FNRGECHHHHHH
EGFRvIll, EVQVLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK
185
W02013185010, GLEVVVSAISGSGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLR
B084-2_HC AEDTAVYYCAGSSGWSEYWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFIDEPVTVSVVNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding to PSMA
E200+PSMA GHNYTTRNILPGLNITSDIVMTQSPSSLSASVGDRVTITCKASQDV 186
binder (nfP2X7 GTAVDWYQQKPGKAPKLLIYWASTRHTGVPDRFTGSGSGTDFTL
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
epitope TISSLQPEDFADYFCQQYNSYPLTFGGGTKLEI KRTVAAPSVFIFP
underlined), PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
US20190300622, VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
B085-1_LC SFNRGECHHHHHH
PSMA, EVQLVQSGAEVKKPGASVKISCKTSGYTFTEYTIHVVVKQASGKGL
187
U320190300622, EWIGNINPNNGGTTYNQKFEDRATLTVDKSTSTAYMELSSLRSED
B085-2_HC TAVYYCAAGWNFDYVVGQGTTVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHHHHH
H
Constructs for binding to CEA
E200+CEA binder GHNYTTRNILPGLNITSDIVLTQSPASLTVSLGLRATISCRASKSVS
188
(nfP2X7 epitope ASGYSYMHVVYQQRPGQPPKLLIYLASNLQSGVPARFSGSGSGT
underlined), DFTLNIHPVEEEDAATYYCQHSRELPTFGGGTKLEIKRTVAAPSVF
W01999043817, IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
B086-1_LC ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKSFNRGECHHHHHH
CEA, EVQLQQSGAELVRSGASVKMSCTASGFNIKDYYMHVVVKQRPEQ
189
W01999043817, GLEWIGWIDPENGDTEYAPKFQGKATMTTDYSSNTAYLQLSSLTS
B086-2_HC EDTAVYYCNTRGLSTMITTRWFFDVWGAGTTVAVSSASTKGPSV
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCHHHHHH
Constructs for binding to PSCA
E200-'-PSCA GHNYTTRNILPGLNITSDIQLTQSPSSLSASVGDRVTITCSASSSV 190
binder (nfP2X7 RFIHVVYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTI
epitope SSLQPEDFATYYCQQVVSSSPFTFGQGTKVEIKRTVAAPSVFIFPP
underlined), SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
US20120077962, TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
B087-1_LC FNRGECHHHHHH
PSCA, EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHVVVRQAPGKG
191
US20120077962, LEWVAWIDPENGDTEFADSVKGRFTISADTSKNTAYLQMNSLRA
B087-2_HC EDTAVYYCKTGGFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHHHHH
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
56
H
Constructs for binding to Lewis Y
E200+Lew is Y GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRSSQRI
192
binder (nfP2X7 VHSNGNTYLEVVYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSG
epitope TDFTFTISSLQPEDIATYYCFQGSHVPFTFGQGTKLQITKRTVAAP
underlined), SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
EP0749482, NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
B088-1_LC SSPVTKSFNRGECHHHHHH
Lewis Y, EVQLVESGGGVVQPGRSLRLSCSSSGFTFSDYYMYVVVRQAPGK
193
EP0749482, GLEVVVAYMSNVGAITDYPDTVKGRFTISRDNSKNTLFLQMDSLRP
B088-2_HC EDTGVYFCARGTRDGSVVFAYWGQGTPVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
Constructs for binding to CD171 Li CAM
E200+CD171_L1 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASQDI
194
CAM binder SNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTL
(nfP2X7 epitope TISSLQPEDFATYFCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFP
underlined), PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
W02008151819, VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
B089-1_LC SFNRGECHHHHHH
CD171_Ll CAM, EVQLVQSGGGLVQSGGSLRLSCRASGYTFTRYVVMLVVVRQRPG
195
W02008151819, HGLEVVVGEINPRNDRTNYNEKFKTRFTISVDRSKSTAYLQMDSLR
B089-2_HC AEDTAVYFCALGGGYAMDYVVGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding to EpCAM
E200+ EpCAM GHNYTTRNILPGLNITSEIVMTQSPATLSVSPGERATLSCRASQSV
196
binder (nfP2X7 SSNLAVVYQQKPGQAPRLIIYGASTTASGIPARFSASGSGTDFTLTI
epitope SSLQSEDFAVYYCQQYNNWPPAYTFGQGTKLEIKRTVAAPSVFIF
underlined), PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
W02010142990A SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
1, B090-1_LC KSFNRGECHHHHHH
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
57
EpCAM, QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISVVVRQAPGQ
197
W02010142990A GLEVVMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSE
1, B090-2_HC DTAVYYCARGLLWNYVVGQGTLVTVSSASTKGPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHHHH
HH
Constructs for binding to ALK
E200+ ALK GHNYTTRNILPGLNITSEIVLTQSPATLSLSPGERATLSCRASESV
198
binder (nfP2X7 DNYGISFAVVYQQKPGQAPRLLIYRASRATGIPARFSGSGSGTDFT
epitope LTISSLEPEDFAVYYCQQNNKDPPTEFGQGTKLEIKRTVAAPSVFI
underlined), FPPSDEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNSQ
W02015069922A ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
2, B091-1_LC TKSFNRGECHHHHHH
ALK, QVQLQQSGAEVKKPGSSVKVSCKASGYAFSSYISVVVRQAPGQG
199
W02015069922A LEWMGGQIYPGDGDTNYAQKFQGRVTITADESTSTAYMELSSLR
2, B091-2_HC SEDTAVYYCVRYYYGSSGYFDYWVVGQGTMVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLOSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCHHHHHH
Constructs for binding to IGF-1R CD221
E200+ IGF-1R GHNYTTRNILPGLNITSDVVMTQTPLSLPVSLGDPASISCRSSQSI
200
binder (nfP2X7 VHSNVNTYLEVVYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGAG
epitope TDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKRTVAAPS
underlined), VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
US798584262, SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
B092-1_LC PVTKSFNRGECHHHHHH
IG F-1 R, QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHVVVKQRPG
201
US798584262, QGLEVVIGEINPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSL
B092-2_HC TSEDSAVYYFARGRPDYYGSSKVVYFDVVVGQGTTVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCHHHHHH
Constructs for binding to Nectin 4
E200+ Nectin 4 GHNYTTRNILPGLNITSSIVMTQTPKFLLVSAGDRVTITCKASQSVS
202
binder (nfP2X7 NDVAVVYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFT
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
58
epitope ISAVQAEDLAVYFCQQDYSSPYTFGGGTKLEI KRTVAA PSVF I FPP
underlined), SD EQ LKSGTASVVC LLN NFYPREAKVQWKVDNALQSGNSQESV
US20210130459, TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
B093-1_LC FNRGECHHHHHH
Nectin 4, QVQLQQSGPELVKPGASVR I SCKASGYTFTTYYI HVVVKQRPGQG
203
U3202101 30459, LEWIGWIYPGNVNTKN N EKFKVKATLTADKSSSTAYMQLSSLTSE
B093-2_HC DSAVYFCARSNPYVMDYWGQGTSVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SG LYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCH
HHHHH
Constructs for binding to FAP
E200+ FAP GHNYTTRN ILPGLN ITSEIVLTQSPGTLSLSPGERATLSCRASQSV
204
binder (nfP2X7 TSSYLAWYQQKPGQAPRLLI NVGSRRATG IPDRFSGSGSGTDFT
epitope LTISRLEPEDFAVYYCQQG IMLPPTFGQGTKVEIKRTVAAPSVF IF P
underlined), PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
EP2603530, VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
13094-1_LC SFNRGECHHHHHH
FAP, EP2603530, EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK
205
B094-2_HC GLEWVSAI IGSGSITYYADSVKGRFTISRDNSKNTLYLQMNSLRAE
DTAVYYCAKGWFGGFNYVVGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVLQS
SG LYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCH
H H HHH
Constructs for binding to AXL
E200+ AXL GHNYTTRN ILPGLN ITSDVVMTQSPLSLPVTLGQPASISC RSSQNI
206
binder (nfP2X7 VHTNGNTYLEVVYQQKPGKAPELLIYKVSNRFSGVPDRFSGSGSG
epitope TDFTLKISRVEAEDVGVYYCFQGSHLLEPFTFGQGTKLEI KRTVAA
underlined), PSVF I F PPSD EQLKSGTASVVCLLNN FYPREAKVQWKVDNALQS
EP2431393, GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
B095-1_LC LSSPVTKSFN RG ECHH H H HH
AXL, EP2431393, QVTLKESG PVLVKPTETLTLTCTVSG FSLSSFGVDVVVRQAPG KG
207
B095-2_HC LEVVMGVIWGGGSTNYNSALKSRLTISKDNSKSQVVLTMTN MDPV
DTATYYCAGEGSKYGAWFA'YVVGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPK
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
59
SCHHHHHH
Constructs for binding to CD138
E200+ C D 138 GHNYTTRN ILPGLN ITSD IQ
MTQSTSSLSASLGDRVTISCSASQGI 208
binder (nfP2X7 NNYLNWYQQKPDGTVELLIYYTSTLQSGVPSRFSGSGSGTDYSL
epitope TISNLEPEDIGTYYCQQYSKLPRTFGGGTKLEIKRTVAAPSVFIFPP
underlined), SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
US20090175863, TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
B096-1_LC FNRGECHHHHHH
CD138, QVQLQQSGSELMMPGASVKISCKATGYTFSNYVVIEWVKQRPGH
209
U5200901 75863, GLEVVIGEILPGTGRTIYNEKFKGKATFTADISSNTVQMQLSSLTSE
B096-2_HC DSAVYYCARRDYYGNFYYAMDYVVGQGTSVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE
PKSCHHHHHH
Constructs for binding to CLDN6
E200+ CLDN6
GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRISENIY 210
binder (nfP2X7 SYLAWYQQKPGKAPKLLIYNAKILVEGVPSRFSGSGSGTDFTLTIS
epitope SLQPEDFATYYCQHHYTVPWWGQGTKLEIKRTVAAPSVFIFPPS
underlined), DEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNSQESVT
W02019056023, EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
B097-1_LC NRGECHHHHHH
CLDN6, EVQLLESGGGLVQPGGSMRLSCAASGFTFSNYWMNVVVRQAPG
211
W02019056023, KGLEWVAQIRLKSDNYATHYADSVKGRFTISRDDSKNTVYLQMN
B097-2_HC SLRAEDTGVYYCNDGPPSGYVVGQGTLLTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding to Her4
E200+ Her4 GHNYTTRNILPGLNITSQSVLTQPASVSGSPGQSITISCAGTSSDV
212
binder (nfP2X7 GGSSYVSVVYQQHPGKAPKLMIYYDSYRPSGVSNRFSGSKSGNT
epitope ASLTISGLQAEDEADYYCSSNTYYSTRVFGGGTKLAVLGKRTVAA
underlined), PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
W02021116119, GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
B098-1_LC LSSPVTKSFNRGECHHHHHH
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
Her4, EVQLVESGGSLVKPGGSLRLSCAASGFTFSNYYMNWVRQAPGK
213
W02021116119, GLEVVISSIDGSSRYIDYADFVKGRFTISRDNATNSLYLQMNSLRAE
B098-2_HC DTAVYYCVRSSSDYFGGGMDVVVGRGTLVTVSSASTKGPSVFPL
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCHHHHHH
Constructs for binding to Claudin 18.2
E200+ Claud in
GHNYTTRNILPGLNITSDIVMTQSPSSLTVTAGEKVTMSCKSSQSL 214
18.2 binder LNSGNQKNYLTVVYQQKPGQPPKLLIYWASTRESGVPDRFTGSG
(nfP2X7 epitope SGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKRTVAA
underlined), PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
SG10201609510 GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
U, B099-1_LC LSSPVTKSFNRGECHHHHHH
Claudin 18.2, QVQLQQPGAELVRPGASVKLSCKASGYTFTSYVVININVKQRPGQ
215
SG10201609510 GLEVVIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTS
U, B099-2_HC EDSAVYYCTRSVVRGNSFDYVVGQGTTLTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding to 0-acetylated GD2
E200+ 0- GHNYTTRNILPGLNITSDVVMTQSPLSLPVTLGQPASISCRSSQSL
216
acetylated G02 LKNNGNTFLHVVYQQRPGQSPRLLIYKVSNRLSGVPDRFSGSGSG
binder (nfP2X7 TDFTLKISRVEAEDVGVYFCSQSTHIPYTFGGGTKVEIKRTVAAPS
epitope VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
underlined), SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
EP3269739A1, PVTKSFNRGECHHHI-IHH
B100-1_LC
0-acetylated EVQLVESGGGLVQPGRSLRLSCTTSEFTFTDYYMTVVVRQAPGK 217
GD2, GLEVVLGFIRNRANGYTTEYNPSVKGRFTISRDNSKSILYLQMNSL
EP3269739A1, KTEDTAVYYCARVSNINAFDYINGQGTLVTVSSASTKGPSVFPLAP
B100-2_HC SSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding to GD3
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
61
E200+ GD3 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCSASQDI
218
binder (nfP2X7 SNYLNVVYQQKPDKAVKLLIFYSSNLHSGVPSRFSGGGSGTDYTL
epitope TISSLQPEDIATYFCHQYSKLPVVTFGQGTKVEIKRTVAAPSVFIFP
underlined), PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
US7253263, VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
B101-1_LC SFNRGECHHHHHH
GD3, EVQLVESGGDFVQPGGSLRVSCAASGFAFSHYAMSVVVRQAPGK
219
US7253263, GLEVVVAYISSGGSGTYYSDSVKGRFTISRDNSKNTLYLQMRSLR
B101-2_HC AEDSAVYFCTRVKLGTYYFDSVVGQGTLLTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
Constructs for binding to GM2
E200+ GM2 GHNYTTRNILPGLNITSDIQLTQSPSSLSASPGDRVTITCSASSSVS
220
binder (nfP2X7 YMHWFQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTSYSLTI
epitope SRLQPEDIATYYCQQRSSYPYTFGGGTKVEIKRTVAAPSVFIFPPS
underlined), DEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNSQESVT
U55939532, EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
B102-1_LC NRGECHHHHHH
GM2, EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDVVVRQAPGQ
221
US5939532, GLEVVMGYIYPNNGGTGYNQKFKSKVTITVDTSTSTAYMELHSLR
B102-2_HC SEDTAVYYCATYGHYYGYMFAYWGQGTLVTVSSASTKGPSVFPL
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCHHHHHH
Constructs for binding to TM4SF1
E200+ TM4SF1 GHNYTTRNILPGLNITSAVVMTQTPLSLPVSLGDQASISCRSSQSL
222
binder (nfP2X7 VHSNGNTYLHVVYMQKPGQSPKVLIYKVSNRFSGVPDRFSGSGS
epitope GTDFTLKISRVEADDLGIYFCSQSTHIPLAFGAGTKLELKRTVAAP
underlined), SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
W02015054427A NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
1, B103-1_LC SSPVTKSFNRGECHHHHHH
TM4SF1, EVILVESGGGLVKPGGSLKLSCAASGFTFSSFAMSVVVRQTPEKR
223
W02015054427A LEWVATISSGSIYIYYTDGVKGRFTISRDNAKNTVHLQMSSLRSED
1, B103-2_HC TAMYYCARRG IYYGYDGYAMDYVVGQGTSVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
CA 03211323 2023- 9-7

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62
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE
PKSCHHHHHH
Constructs for binding to C0147
E200+ C D147 GHNYTTRN ILPGLN ITSD IQ
MTQSPSTLSASVGDRVTLSCKASENV 224
binder (nfP2X7 GTYVSWYQQKPGKAPKLLIYGASNRYTGVPSRFTGSGSGTDFTL
epitope TISSLQPEDFATYYCGQSYSYPFTFGSGTKLEIKRTVAAPSVFIFP
underlined), PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
W02017186182, VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
B104-1_LC SFNRGECHHHHHH
CD147, EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFWMNWVRQAPG
225
W02017186182, KGLEWVSEIRLKSNNYATHYAESVKGRFTISRDDSKNTLYLQMNS
B104-2_HC LKTEDTAVYYCTSYDYEYVVGQGTLVTVSAASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCH
HHHHH
Constructs for binding to CEACAM5
E200+ CEACAM5 GHNYTTRNILPGLNITSDIQLTQSPSSLSASVGDRVTITCKASQDV
226
binder (nfP2X7 GTSVAWYQQKPGKAPKLLIYANTSTRHTGVPSRESGSGSGTDFTF
epitope TISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKRTVAAPSVFIFPPS
underlined), DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
W02015069430, EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
B105-1_LC NRGECHHHHHH
CEACAM5, EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYVVMSVVVRQAPG 227
W02015069430, KGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRP
B105-2_HC EDTGVYFCASLYFGFPWFAYWGQGTPVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding to VEGFR-1
E200+ VEGFR-1 GHNYTTRNILPGLNITSDIVMTQSPDSLAVSLGERATINCSASSSV
228
binder (nfP2X7 SYMHVVYQQKPGQPPKLLIYRTSNLASGVPDRFSGSGSGTDFTLT
epitope ISSLQAEDVAVYYCHQWSMYTFGQGTKVEIKRTVAAPSVFIFPPS
underlined), DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
US7615214, EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
63
B106-1_LC NRGECHHHHHH
VEGFR-1, QVQLVQSGAEVKKPGASVKVSCKASGYTFINYNMHWVRQAPGQ
229
US7615214, GLEVVMGAIFPGNGFTSYNQKFKGRVTITVDKSTSTAYMELSSLRS
B106-2_HC EDTAVYYCARDGDYYFDYVVGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCH
HHHHH
Constructs for binding to Podoplanin (PDPN)
E200+ PDPN
GHNYTTRNILPGLNITSDVLMTQTPLSLPVSLGDQASISCRSSRNI 230
binder (nfP2X7 VQSTGNTYLEWYLQKPGQSPKLLIFKVSNRFSGVPDRFSGSGSG
epitope TDFTLKISRVEAEDLGVYYCFQGSHVPPVVTEGGGTKLEIKRTVAA
underlined), PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
US20160347834 GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
Al, B107-1_LC LSSPVTKSFNRGECHHHHHH
PDPN, DVQLVESGGGLVQPGGSRKLSCAASGFTFSGFGMHWVRQAPEK
231
US20160347834 GLEVVVAYISSVSSRIYYADTVKGRFTISRDNPKNTLFLQMTSLRSE
Al, B107-2_HC DTAMYYCAREQTGPAVVFAYWGQGTLVTVSAASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding to VVT1
E200+ VVT1 GHNYTTRNILPGLNITSQTVVTQPPSASGTPGQRVTISCSGSSSNI
232
binder (nfP2X7 GSNYVYWYQQLPGTAF'KLVLLIYRSNQRPSGVPDRFSGSKSGTS
epitope ASLAISGPRSVDEADYYCAAVVDDSLNGVVFGGGTKLTVLGKRTV
underlined), AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ
EP2694553A2, SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ
B108-1_LC GLSSPVTKSFNRGECHHHHHH
VVT1, QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISVVVRQAPGQ
233
EP2694553A2, GLEVHWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLR
B108-2_HC SEDTAVYYCARRIPPYYGMDVWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
Constructs for binding to GPC2
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
64
E200+ GPC2 GHNYTTRNILPGLNITSENVLTQSPAIMSASLGEKVTMSCRASSSV
234
binder (nfP2X7 NYIYVVYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTI
epitope SSMEGEDAATYYCQQFSSSPSTFGTGTKLELKRTVAAPSVFIFPP
underlined), SDEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNSQESV
W02020033430, TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
B109-1_LC FNRGECHHHHHH
GPC2, EVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIHVVVKQSPGKT
235
W02020033430, LEWIGYINPNNGDIFYKQKFNGKATLTINKSSNTAYMELRSLTSED
B109-2_HC SAVYYCVRSSNIRYTFDRFFDVVVGTGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
Constructs for binding to FGFR4
E200+ FGFR4 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASESV
236
binder (nfP2X7 STLMHVVYQQKPGKAPKLLIYGTSNLESGVPSRFSGSGSGTDFTL
epitope TISSLQPEDFATYYCQQSWNDPPTFGGGTKVEIKRTVAAPSVFIF
underlined), PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
W02019034427A SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
1, B110-1_LC KSFNRGECHHHHHH
FGFR4, EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGK
237
W02019034427A GLEVVVATINPSGTRTYYPDSVKGRFTISRDNAKNSLYLQMNSLRA
1, B110-2_HC EDTAVYYCARLYNNYAFDYWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVN H KPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding to EphB4
E200+ EphB4 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASQDV
238
binder (nfP2X7 STAVAVVYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT
epitope ISSLQPEDFATYYCQQTAQTPETFGQGTKVEIKRTVAAPSVFIFPP
underlined), SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
EP1973950, TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
B111-1_LC FNRGECHHHHHH
EphB4, EVQLVESGGGLVQPGGSLRLSCAASGFTISGYYIHVVVRQAPGKG
239
EP1973950, LEWVGGIYLYSGSTDYADSVKGYADSVKGRFTISADTSKNTAYLQ
Bill -2_HC MNSLRAEDTAVYYCARGSGLRLGGLDYAMDYWGQGTLVTASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCHHHHHH
Constructs for binding to STEAP-1
E200+ STEAP-1 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCKSSQSL
240
binder (nfP2X7 LYRSNQKNYLAVVYQQKPGKAPKLLIYVVASTRESGVPSRFSGSGS
epitope GTDFTLTISSLQPEDFATYYCQQYYNYPRTFGQGTKVEIKRTVAA
underlined), PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
W02015112909, GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
B112-1_LC LSSPVTKSFNRGECHHHHHH
STEAP-1, EVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAVVNVVVRQAPG
241
W02015112909, KGLEWVGYISNSGSTSYNPSLKSRFTISRDTSKNTLYLQMNSLRA
B112-2_HC EDTAVYYCARERNYDYDDYYYAMDYVVGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCHHHHHH
Constructs for binding to STEAP-2
E200+ STEAP-2 GHNYTTRNILPGLNITSDIQMTQSPSTLSASVGDRVTITCRASQSIS
242
binder (nfP2X7 SWLAVVYQQKPGRAPNLLISKASSLKSGVPSRFSGSGSGTEFTLT
epitope VSSLQPDDFATYYCQQYYSYSYTFGQGTKLEIKRTVAAPSVFIFP
underlined), PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
US20180104357, VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
B113-1_LC SFNRGECHHHHHH
STEAP-2, QVQLVESGGGVVQPGRSLRLSCVASGFTISSYGMNVVVRQAPGK
243
US20180104357, GLEWVAVISYDGGNKYSVDSVKGRFTI SRDNSKNTLYLQMNSLR
B113-2_HC AEDSAVYYCARGRYFDLWGRGTLVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHH
HHHH
Constructs for binding to IL11Ra
E200+ IL11Ra
GHNYTTRNILPGLNITSDIVLTQSPATLSMTPGDSVSLSCRASQSI 244
binder (nfP2X7 SNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLS
epitope FNSVETEDFGVYFCQQRYSWPLTFGAGTKLEMKRTVAAPSVFIF
underlined), PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
W02018109170, SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
66
B114-1_LC KSFNRGECHHHHHH
IL11Ra, QVQLQQPGAELVRPGSSVKLSCKASGYTFTNYVVMHWLKQRPVQ
245
W02018109170, GLEWIGNIGPSDSKTHYNQKFKDKATLTVDKSSSTAYMQLNSLTS
B114-2_HC EDSAVYYCARGDYVLFTYVVGQGTLVTVSAASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCH
HHHHH
Constructs for binding to CD163
E200+ CD163 GHNYTTRNILPGLNITSDIVMTOSPSSLSASVGDRVTITCRASQSV
246
binder (nfP2X7 SSDVAWFQQKPGKSPKPLIYYASNRYSGVPSRFSGSGSGTDFTL
epitope TISSLQAEDFAVYFCGQDYTSPRTFGGGTKLEIKRTVAAPSVFIFP
underlined), PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
W02011039510A VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
2, B115-1_LC SFNRGECHHHHHH
CD163, QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQFPGN
247
W02011039510A KLEWMGYITYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAA
2, B115-2_HC DTATYYCVSGTYYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKS
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHHH
HHH
Constructs for binding to Chlorotoxin
E200+ CLTX GHNYTTRNILPGLNITSMCMPCFTTDHQMARKCDDCCGGKGRG
248
binder (nfP2X7 KCYGPQCLCRKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP
epitope REAKVQVVKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD
underlined), YEKHKVYACEVTHQGLSSPVTKSFNRGECHHHHHH
W02017066481A
1, B116-1_LC
CLTX, MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCRASTKGP
249
W02017066481A SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
1, B116-2_HC HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
KKVEPKSCHHHHHH
Constructs for binding to CD206 Nanobody VH
E200+ CD206 GHNYTTRNILPGLNITSQVQLQESGGGLVQPGGSLRLSCAASGFS
250
binder (nfP2X7 LDYYAIGWFRQAPGKEREGISCISYKGGSTTYADSVKGRFTISKD
CA 03211323 2023- 9-7

WO 2022/187906
PCT/AU2022/050206
67
epitope NAKNTAYLQMNSLKPEDTGIYSCAAGFVCYNYDYWGQGTQVTV
underlined), SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
W02014140376A SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
1, B117-2_HC KPSNTKVDKKVEPKSCHHHHHH
Constructs for binding to IL1RAP
E200+ IL1RAP GHNYTTRNILPGLNITSDVQMTQSPSSLSASVGDRVTITCQASQSI
251
binder (nfP2X7 YSFLSWYQQKPGQAPKLLIYAASDLESGVPSRFSGSGSGTDFTLT
epitope ISSLQPEDFATYYCQCNYIIDYGAFGQGTKVVIKRTVAAPSVFIFPP
underlined), SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
W02017191325A TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
9, B118-1_LC FNRGECHHHHHH
IL1RAP, EVQLEESGGRLVQPGTSLRLSCAVSGFSLSSYDMSWVRQAPGK
252
W02017191325A GLEVVVSTIYIGGTTAYASWPKGRFTISKTNSKNTLYLQMNSLRAE
9, B118-2_HC DTAVYFCARLQGANYYNSLALWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
Constructs for binding to MICA
E200+ MICA GHNYTTRNILPGLNITSAIQLTQSPSSLSASVGDRVTITCRASQGIS
253
binder (nfP2X7 SALAWYQQKPGKVPKSLIYDASSLESGVPSRFSGSGSGTDFTLTI
epitope SSLQPEDFATYYCQQFNSYPITFGQGTRLEIKRTVAAPSVFIFPPS
underlined), DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
W02019183551, EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
B119-1_LC NRGECHHHHHH
MICA, QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVRQAPGE
254
W02019183551, GLEVVVALIVVYDGSNKFYGDSVKGRFTISRDNSKNTLYLQMNSLS
B119-2_HC AEDTAVYYCAREGSGHYVVGQGTLVTVSSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHH
HHHH
Constructs for binding to MAGE-Al scTcR
E200+ MAGE-A1 GHNYTTRNILPGLNITSMKPTLISVLVIIFILRGTRAQRVTQPEKLLS
255
binder (nfP2X7 VFKGAPVELKCNYSYSGSPELFVVYVQYSRQRLQLLLRHISRESIK
epitope GFTADLNKGETSFHLKKPFAQEEDSAMYYCALRSGGYQKVTFGT
underlined), GTKLQVIPGGGGSGGGGSGGGGSMGIRLLCRVAFCFLAVGLVDV
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
68
AU2018234830A KVTQSSRYLVKRTGEKVFLECVQDMDHENMFVVYRQDPGLGLRLI
1, B120-1_LC
YFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLC
ASNNRDSYNSPLHFGNGTRLTVTHHHHHH
Constructs for binding to MAGE-Al sTCR
E200+ MAGE-A1 GHNYTTRNILPGLNITSMKPTLISVLVIIFILRGTRAQRVTQPEKLLS 256
binder (nfP2X7 VFKGAPVELKCNYSYSGSPELFVVYVQYSRQRLQLLLRHISRESIK
epitope
GFTADLNKGETSFHLKKPFAQEEDSAMYYCALRSGGYQKVTFGT
underlined),
GTKLQVIPIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSK
AU2018234830A DSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSI IP
1, B121-1_alpha EDTFFPSPESS
chain
MAGE-A1,
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQD 257
AU2018234830A MDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREK
1, B121-2_beta KERFSLILESASTNQTSMYLCASNNRDSYNSPLHFGNGTRLTVTD
chain LKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSVWVV
NGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFVVQDP
RNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAVVGRADHHH
HHH
Constructs for binding to MAGE-Al sTCR/2
E200+ MAGE-A1 MKPTLISVLVIIFILRGTRAQRVTQPEKLLSVFKGAPVELKCNYSYS 258
binder (nfP2X7
GSPELFVVYVQYSRQRLQLLLRHISRESIKGFTADLNKGETSFHLK
epitope KPFAQEEDSAMYYCALRSGGYQKVTFGTGTKLQVIPIQNPDPAVY
underlined),
QLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRS
AU2018234830A MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSGGGG
1, B122-1_alpha SGHNYTTRNILPGLNITS
chain
MAGE-A1,
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQD 259
AU2018234830A MDHENMFVVYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREK
1, B122-2_beta KERFSLILESASTNQTSMYLCASNNRDSYNSPLHFGNGTRLTVTD
chain LKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSVWVV
NGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFVVQDP
RNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAVVGRADHHH
HHH
Constructs for binding TRBC1
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
69
E200+TRBC1 GHNYTTRNILPGLNITSDWMTQSPLSLPVSLGDQASISCRSSQRL 260
binder (nfP2X7 VHSNGNTYLHVVYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSG
epitope TDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKRTVAAPS
underlined), Jovi- VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGN
1, SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
W02015132598A PVTKSFNRGECHHHHHH
1, B122-1_LC
TRBC1, Jovi-1, EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHVVVKQRPGQ
261
W02015132598A GLEVVIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSE
1, B122-2_HC DSAVYYCARGAGYNFDGAYRFFDFVVGQGTTLTVSSASTKGPSV
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCHHHHHH
Constructs for binding TRBC2
E200+TRBC2 GHNYTTRNILPGLNITSQPQSVSESPGKTVTISCTRSSGNFASKYV 262
binder (nfP2X7 QWYQQRPGSSPTTVIYENYQRPSGVPDRFSGSIDSSSNSATLTIS
epitope GLKTEDEADYYCQSYDEVSWFGGGTQLTVLGQPAAKRTVAAPS
underlined), F09, VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGN
W02015132598A SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
1, B123-1_LC PVTKSFNRGECHHHHHH
TRBC2, F09,
QMQLVQSGAEVKKPGASVKVSCKASGYTFASYYHVVVRQAPGQ 263
W02015132598A GLEVVGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSRLRS
1, B123-2_HC DDTAVYYCASNRGGSYKSVGMDVVVGQGTTVIVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCHHHHHH
Constructs for binding urokinase-type plasminogen activator receptor (u PAR)
E200+uPAR GHNYTTRNILPGLNITSDIVLTQSPASLAVSLGQRATISCRASKSVS 264
binder (nfP2X7 TSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTD
epitope FTLDIHPVEEEDAATYYCQHSRELPYTFGGGTKLELKRTVAAPSV
underlined), 7G1, FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNS
W02006094828, QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
B124-1_LC VTKSFNRGECHHHHHH
uPAR, 7G1, VQLQESGPELKKPGETVKISCKASGYTFTDYSMHVVVKQAPGKGL
265
W02006094828, KCMGVVINTETTKSTYADDFKGRFALSLETSASTVYLQISNLKNED
TATYFCAREASYGEFDYWGQGTTVTVSSASTKGPSVFPLAPSSK
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
B124-2_HC
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHH
HHHH
EGFRvl I I targeted CAR
EGFRvIl I targeted EVQVLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK 266
CAR scFv_(HL- GLEVVVSAISGSGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLR
configuration_clon AEDTAVYYCAGSSGWSEYWGQGTLVTVSSGGGGSGGGGSGG
e_139)_CD8a_C GGSDIQMTQSPSSLSASVGDRVTITCRASQGIRNNLAVVYQQKPG
D8TM_CD28_CD KAPKRLIYAASNLQSGVPSRFTGSGSGTEFTLIVSSLQPEDFATYY
137_CD3zeta
CLQHHSYPLTSGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEA
CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSK
RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRK
KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS
ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL
STATKDTYDALHMQALPPR
EGFRvl II peptide LEEKKGNYVVTDH
267
Constructs for binding CD33
EGFRvIl I-
LEEKKGNYVVTDHDIQLTQSPSTLSASVGDRVTITCRASESLDNY 268
peptide+C033 GIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFT
binder (EGFRvl II LTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVAAPSVFI
epitope
FPPSDEQLKSGTASVVCLLNNFYPREAKVQINKVDNALQSGNSQ
underlined)
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV
TKSFNRGECHHHHHH
CD33,
Gemtuzumab,
B030-3_LC
CD33,
EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHVVVRQAPGQS .. 269
LEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSE
Gemtuzumab,
DTAFYYCVNGNPWLAYVVGQGTLVTVSSASTKGPSVFPLAPSSKS
13030-2_HC
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHHH
HHH
Constructs for binding Her2
EGFRvIl I-
LEEKKGNYVVTDHDIQMTQSPSSLSASVGDRVTITCRASQDVNTA 270
CA 03211323 2023- 9-7

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71
peptide+Her2 VAVVYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISS
binder (EGFRvIll LQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSD
epitope EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
underlined) QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN
RGECHHHHHH
Her2,
Gemtuzumab,
B033-3_LC
Her2, EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVVVRQAPGKG
271
LEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAE
Trastuzumab,
DTAVYYCSRWGGDGFYAMDYVVGQGTLVTVSSASTKGPSVFPLA
B033-2_HC
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
CLDN6 targeted CAR
CLDN6 targeted EVQLLESGGGLVQPGGSMRLSCAASGFTFSNYVVMNVVVRQAPG
272
KGLEWVAQIRLKSDNYATHYADSVKGRFTISRDDSKNTVYLQMN
CAR scFv_(HL-
SLRAEDTGVYYCNDGPPSGYVVGQGTLLTVSSGGGGSGGGGSG
configuration_clon
GGGSDIQMTQSPSSLSASVGDRVTITCRISENIYSYLAVVYQQKPG
e_Ab3- KAPKLLIYNAKILVEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
4)_CD8a_CD8TM QHHYTVPVVTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEA
CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSK
¨CD28¨CD137¨C RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRK
D3zeta KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS
ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL
STATKDTYDALHMQALPPR
CLDN6 peptide 23- VVTAHAIIRDFYNPLVAEAQKREL
273
mer
CLDN6 peptide 13- TAHAIIRDFYNPL
274
mer
CLDN6 peptide 10- LVAEAQKREL
275
mer
Constructs for binding C033
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
72
CLDN6- VVTAHAIIRDFYNPLVAEAQKRELDIQLTQSPSTLSASVGDRVTITC
276
peptide+C033 RASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFS
binder (CLDN6 GSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK
epitope RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQVVKVDN
underlined) ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
HQGLSSPVTKSFNRGECHHHHHH
CD33,
Gemtuzumab,
B030-4_LC
C033, EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHVVVRQAPGQS
277
LEVVIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSE
Gemtuzumab,
DTAFYYCVNGNPWLAYVVGQGTLVTVSSASTKGPSVFPLAPSSKS
B030-2_HC
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHHH
HHH
Constructs for binding Her2
CLDN6- VVTAHAIIRDFYNPLVAEAQKRELDIQMTQSPSSLSASVGDRVTITC
278
peptide+Her2 RASQDVNTAVAVVYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRS
binder (CLDN6 GTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAP
epitope SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
underlined) NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGECHHHHHH
Her2,
Trastuzumab,
B033-4_LC
Her2, EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVVVRQAPGKG
279
LEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAE
Trastuzumab,
DTAVYYCSRWGGDGFYAMDYVVGQGTLVTVSSASTKGPSVFPLA
B033-2_HC
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCHHHHHH
Constructs for binding B7-H7 (HHLA2)
E200+B7H7 GHNYTTRNILPGLNITSDIVMTQSPSSLAVSAGEKVTISCLSSQSLF 280
binder (nfP2X7 SSNTKRNYLNWYLQKPGQSPKLLIYHASTRLTGVPGRFIGSGSGT
DFTLTVSTVQAEDLGDYFCQQHYETPLTFGDGTRLEIKRTVAAPS
epitope
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
CA 03211323 2023- 9-7

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PCT/AU2022/050206
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underlined), 4.5, SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
W02014100823A PVTKSFNRGECHHHHHH
1, B125-1_LC
67H7, 4.5, QIQLQESGPGLVKPSQSLSLTCSVTGFSITTGGYYWNINIRQFPGK
281
W02014100823A KLEWMGYIYTSGRTSYNPSLKSRISITRDTSKNQFFLQLNSMTTE
DTATYYCADMADKGGWFAYWGQGTLVTVSSASTKGPSVFPLAP
1, B125-2_HC
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding C034
E200+CD34 GHNYTTRNILPGLNITSDVLLTQSPLSLPVTLGQPASISCRSSQTIV 282
HSNGNTYLEWFQQRPGQSPRLLIYQVSNRFSGVPDRFSGSGSG
binder (nfP2X7
TDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKRTVAAP
epitope
SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
underlined), NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
h4C8b SSPVTKSFNRGECHHHHHH
,
US20100311955
Al, B126-1_LC
0D34, h4C8b, QIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQ 283
US20100311955 GLKWMGWINTNTGEPKYAEEFKGRFALSLDTSVSTAYLQINSLKA
EDTAVYFCARGYGNYARGAWLAYWGQGTLVTVSSASTKGPSVF
Al, B126-2_HC
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCHHHHHH
Constructs for binding 007
E200+CD7 binder GHNYTTRNILPGLNITSDIQMTQTTSSLSASLGDRVTISCSASQGIS 284
NYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTI
(nfP2X7 epitope
SNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRTVAAPSVFIFPPS
underlined), seq
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
1, EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
W02003051926, NRGECHHHHHH
B127-1_LC
CA 03211323 2023- 9-7

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CD7, seq 1,
EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSVVVRQTPEK 285
W02003051926, RLEVVVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSE
DTAMYYCARDEVRGYLDVWGAGTTVTVSSASTKGPSVFPLAPSS
B127-2_HC
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCH
HHHHH
Constructs for binding CD7
E200+CD7 binder GHNYTTRNILPGLNITSMDVQLQESGGGSVQAGGSLRLSCPASG 286
YTFSHYCMGWNRQAPGKEREEVATIDTDDTPTYADSVMGRFTIS
(nfP2X7 epitope
RDNANNALYLQMNDLKPEDTSMYYCAIWMKLRGSCHDRRLEVR
underlined), seq
GQGTQVTVSINASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
32,
EPVTVSVVNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
US20170226204, QTYICNVNHKPSNTKVDKKVEPKSCHHHHHH
B128-1_HC
Constructs for binding GPRC5D
E200+GPRC5D GHNYTTRNILPGLNITSQSVLTQPPSVSAAPGQKVTIPCSGSRSN 287
binder (nfP2X7 VGNYYVSVVYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSA
TLGITGLQTGDEADYFCGTVVDGSLSAHVFGTGTKVTVLGRTVAA
epitope
PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
underlined), seq GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
1 LSSPVTKSFNRGECHHHHHH
,
SG10202007326
Q, B129-1_LC
GPRC5D, seq 1, QVQLVQSGSELKKPGASVRVSCTASGYTFTSYYMHVVVRQAPGQ
288
GLEVVMGVINPNAGSTRYAQKFQGRVTMSTDTSTSTAYMDLSSLR
SG10202007326
SEDTAVYYCARGMYRSLLFYDPWGQGTLVTVSSASTKGPSVFPL
Q, B129-2_HC APSSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCHHHHHH
Constructs for binding TIM-3
E200+TIM-3 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCLASQPIG 289
IWLAVVYQQKPGKAPKLLIYAATSLADGVPSRFSGSGSGTDFTFTI
binder (nfP2X7
SSLQPEDIATYYCQQLYSSPWTEGGGTKVEIKRTVAAPSVFIFPPS
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
epitope DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
underlined) EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
,
NRGECHHHHHH
h1701-009NKG,
EP3587452,
B130-1_LC
TIM-3, h1701- QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNVVVRQAPGQ
290
009NKG GLEVVMGDIIPNKGGSKYNQKFKDRVTMTTDTSTSTAYMELRSLR
,
SDDTAVYYCATVVGYGSSYRWFDYWGQGTLVTVSSASTKGPSVF
EP3587452,
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
B130-2_HC PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCHHHHHH
Constructs for binding to CD191 (CCR1)
E200+CD191 GHNYTTRNILPGLNITSDIVMTOSPLSLPVTLGEPASISCRSSQSLV 291
HRNGITFFHWYLQKPGQSPKLLIYKISNRFSGVPDRFSGSGSGTD
binder (nfP2X7
FTLKISRVEAEDVGVYFCSQGTHVPPTFGQGTKLEIKRTVAAPSV
epitope
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQVVKVDNALQSGNS
underlined), QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGECHHHHHH
hzmAb5-
06_LV5HV14,
EP3656791,
B131-1_LC
CD191, hzmAb5- QVQLQQSGPGLVKPSQTLSITCTVSGFSLNNYGVHVVVRQPPGK
292
LV5HV14 GLEVVLGVIWSAGTTVYNAAAISRLTISKDTSKNQVSFKMSSLTAA
06 _ ,
DTAVYYCAKDGSRYYTAMDYVVGQGTLVTVSSASTKGPSVFPLAP
EP3656791,
SSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFPAVL
B131-2_HC QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding to CD66b (CEACAM8)
E200+CD66b GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCRASSSV 293
SYMHINYQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFTFTI
binder (nfP2X7
SSLQPEDIATYYCQQWSSNPLTFGQGTKVEIKRTVAAPSVFIFPPS
epitope
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
underlined), EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
CA 03211323 2023- 9-7

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BW250-183, NRGECHHHHHH
EP0585570A1,
B132-1_LC
CD66b, BVV250- QVQLQESGPGLVRPSQTLSLTCTVSGFSDYYMNVVVRQPPGRGL
294
183 EWIGFISNKPNGHTTEYSASVKGRVTMLRDTSKNQFSLRLSSVTA
,
ADTAVYYCARDKGIRVVYFDVVVGQGSLVTVSSASTKGPSVFPLAP
EP0585570A1' SSKSTSGGTAALGCLVKDYPPEPVTVSWNSGALTSGVHTFPAVL
B132-2_HC QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS
CHHHHHH
Constructs for binding to CD11 b (MAC-1)
E200+CD66b GHNYTTRNILPGLNITSDIQMTQSPSSLSASLGERVSLTCRASQEI 295
SGYLSWHQQKPDGTIKRLLYSTSTLDSGVPKRFSGSRSGSDYSL
binder (nfP2X7
TISSLESEDFADYYCLQYAISPPTFGGGTKLEIKRTVAAPSVFIFPP
epitope
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
underlined), TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
FNRGECHHHHHH
seq_1,
W0201 7220369A
1, B133-1_LC
CD11 b, seq_1, QVTLKESGPGILQTSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGK
296
GLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTTD
W02017220369A
TATYYCALNYYNSTYNFDFWGQGTTLTVSSASTKGPSVFPLAPS
1, B133-2_HC
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
HHHHHH
Constructs for binding to EMR2 (ADGRE2)
E200+EMR2 GHNYTTRNILPGLNITSDIQMTQSPSSLSASVGDRVTITCKASQNV 297
binder (nfP2X7 RTTVDWYQQKPGKAPKLLIYLASNRHTGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCLQHRNYPLTFGGGTKVEIKRTVAAPSVFIFP
epitope
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
underlined), VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
hSC93.253, SFNRGECHHHHHH
W0201 7087800A
CA 03211323 2023- 9-7

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1, B134-1_LC
EMR2,
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGK 298
hSC93.253, GLEVVVSTISSGGNYNYYPDSVKGRFTISRDNAKNSLYLQMNSLR
AEDTAVYYCARHYDYPDYAMDYVVGQGTTVTVSSASTKGPSVFP
W02017087800A
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSVVNSGALTSGVHTFP
1, B134-2_HC AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE
PKSCHHHHHH
Constructs for binding to MUC16
E200+MUC16 GHNYTTRNILPGLNITSDIVMTQAAPSVPVTPGESVSISCRSSKSL 299
LHSNGNTYLYVVFLQRPGQSPQRLIYYMSNLASGVPDRFSGRGS
binder (nfP2X7
GTDFTLRISRVEAEDVGVYYCMQSLEYPLTFGGGTKLEIKRTVAA
epitope
PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
underlined),
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
18C6 LSSPVTKSFNRGECHHHHHH
,
W02016149368A
1, B135-1_LC
MUC16, 18C6, QVTLKESGPGILQPSQTLSLTCSFSGFSLSTVGMGVGVVSRQPSG 300
KGLEWLAHIWVVDDEDKYYNPALKSRLTISKDTSKNQVFLKIANVD
W02016149368A
TADTATYYCTRIGTAQATDALDYWGQGTSVTVSSASTKGPSVFPL
1, B135-2_HC
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCHHHHHH
Constructs for binding to NYESO-1 HLA-A2 (scTCR)
E200+ scTCR GHNYTTRNILPGLNITSAQSVAQPEDLVNVAEGNPLTVKCTYSVS 301
NYESO-1 v1 GNPYLFWYVQYPNRGLQFLLKYLGDSALVKGSYGFEAEFNKSQT
SFHLKKPSALVSDSALYFCAVRDINSGAGSYQLTFGKGTKLSVIPG
binder (nfP2X7
GGGSGGGGSGGGGSSAVISQKPSRDIKQRGTSLTIQCQVDKRLA
epitope
LMFVVYRQQPGQSPTLIATAVVTGGEATYESGFVIDKFPISRPNLTF
underlined),
STLTVSNMSPEDSSIYLCSVGGSGAADTQYFGPGTRLTVLHHHH
HH
18C6,
W02017109496A
1, B136-1_v1
CA 03211323 2023- 9-7

WO 2022/187906 PCT/AU2022/050206
78
E200+ scTCR HHHHHHAQSVAQPEDLVNVAEGNPLTVKCTYSVSGNPYLFVVYV .. 302
NYES0-1v2 QYPNRGLQFLLKYLGDSALVKGSYGFEAEFNKSQTSFHLKKPSAL
VSDSALYFCAVRDINSGAGSYQLTFGKGTKLSVIPGGGGSGGGG
binder (nfP2X7
SGGGGSSAVISQKPSRDIKQRGTSLTIQCQVDKRLALMFVVYRQQ
epitope
PGQSPTLIATAVVTGGEATYESGFVIDKFPISRPNLTFSTLTVSNMS
underlined),
PEDSSIYLCSVGGSGAADTQYFGPGTRLTVLGGGGSGHNYTTRN
ILPGLN ITS
W02017109496A
1, B136-2_v2
Constructs for binding to SURVIVIN HLA-A2 (scTCR)
E200+ scTCR
GHNYTTRNILPGLNITSSQTIHQVVPATLVQPVGSPLSLECTVEGTS 303
SURVIVINv1
NPNLYVVYRQAAGRGLELLFYSVGIGQISSEVPQNLFASRPQDRQ
FILSSKKLLLSDSGFYLCAVVSIGAEQFFGPGTRLTVLEDLKNGSAD
binder (nfP2X7
DAKKDAAKKDGKSGGGGSGGGGSGGGGSQKEVEQNSGPLSVP
epitope
EGAIASLNCTYSDRGSQSFFVVYRQYSGKSPELIMSIYSNGDKED
GRFTAQLN KASQYVSLLI RDSQPSDSATYLCAVSKGYKMFGDGT
underlined),
QLVVKPNIHHHHHH
18C6, NZ719707,
B137-1_v1
E200+ scTCR HHHHHHSQTIHQVVPATLVQPVGSPLSLECTVEGTSNPNLYWYR 304
NYES0-1v2
QAAGRGLELLFYSVGIGQISSEVPQNLFASRPQDROFILSSKKLLL
SDSGFYLCAWSIGAEQFFGPGTRLTVLEDLKNGSADDAKKDAAK
binder (nfP2X7
KDGKSGGGGSGGGGSGGGGSQKEVEQNSGPLSVPEGAIASLN
epitope
CTYSDRGSQSFFVVYRQYSGKSPELIMSIYSNGDKEDGRFTAQLN
underlined),
KASQYVSLLIRDSQPSDSATYLCAVSKGYKMFGDGTQLVVKPNIG
GGGSGHNYTTRNILPGLNITS
NZ719707, B137-
2_v2
Constructs for biding to BCMA (ligand)
E200+ dAPRIL GHNYTTRNILPGLNITSSVLHLVPINATSKDDSDVTEVMVVQPALR 305
binder (nfP2X7 RGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREG
QGRQETLFRCI RSMPSHPDRAYNSCYSAGVFHLHQGDILSVI IPR
epitope
ARAKLNLSPHGTFLGFVKLSGGGSDPHHHHHH
underlined),
W02015052538A
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1, B138-1_v1
E200+ dAPRIL HHHHHHSVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQG
306
binder (nfP2X7 YGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRC
IRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG
epitope
TFLGFVKLSGGGSDPGHNYTTRNILPGLNITS
underlined),
W02015052538A
1, B139-2_v2
Constructs for binding to CD200
E200+ CD200, QVQLQQSGSELKKPGASVKISCKASGYSFTDYIILVVVRQNPGKGL
307
EWIGHIDPYYGSSNYNLKFKGRVTITADQSTTTAYMELSSLRSED
samalizumab,
TAVYYCGRSKRDYFDYVVGQGTTLTVSSASTKGPSVFPLAPSSKS
B140-1
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHHH
HHH
CD200, LC,
DIQMTQSPSSLSASIGDRVTITCKASQDINSYLSWFQQKPGKAPK 308
LLIYRANRLVDGVPSRFSGSGSGTDYTLTISSLQPEDFAVYYCLQY
samalizumab,
DEFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
B140-2
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECHHHHHH
Further examples of tumour-specific epitope antigen moieties (including linker
and hinge regions)
E200+IgG hinge GHNYTTRNILPGLNITSEPKSSDKTHT
365
(E200 underlined)
E200+GS
GHNYTTRNILPGLNITSGSEPKSSDKTHT 366
linker_IgG hinge
E200+G4S
GHNYTTRNILPGLNITSGGGGSEPKSSDKTHT 367
linker+IgG hinge
Extended E200+ GHNYTTRNILPGLNITSTFHKTSGSGKEPKSSDKTHT
368
_IgG hinge
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Extended E200+ GHNYTTRNILPGLNITSTFHKTSGSGKGSEPKSSDKTHT
369
GS linker+_IgG
hinge
Extended E200+ GHNYTTRNILPGLNITSTFHKTSGSGKGGGGSEPKSSDKTHT
370
G4S linker+IgG
hinge
E200+IgG GHNYTTRNILPGLNITSEPKSSDKTHTGS
371
hinge-1-GS linker
E200+GS GHNYTTRNILPGLNITSGSEPKSSDKTHTGS
372
linker+IgG
hinge+GS linker
E200+G4Slinker+ GHNYTTRNILPGLNITSGGGGSEPKSSDKTHTGS
373
IgG hinge+GS
lin ker
Extended GHNYTTRNILPGLNITSTFHKTSGSGKEPKSSDKTHTGS
374
E200+_IgG
hinge+GSlinker
Extended GHNYTTRNILPGLNITSTFHKTSGSGKGSEPKSSDKTHTGS
375
E200+GS
linker+ _IgG
hinge+GSlinker
Extended GHNYTTRNILPGLNITSTFHKTSGSGKGGGGSEPKSSDKTHTGS
376
E200+G4S
linker+ _IgG
hinge+GSlinker
E200+IgG GHNYTTRNILPGLNITSEPKSSDKTHTGGGGS
377
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81
hinge+G4S linker
E200+GS GHNYTTRNILPGLNITSGSEPKSSDKTHTGGGGS
378
linker+IgG
hinge+G4S linker
E200+G4S GHNYTTRNILPGLNITSGGGGSEPKSSDKTHTGGGGS
379
linker+IgG
hinge+G4S linker
Extended GHNYTTRNILPGLNITSTFHKTSGSGKEPKSSDKTHTGGGGS
380
E200+IgG
hinge+G4S linker
Extended GHNYTTRNILPGLNITSTFHKTSGSGKGSEPKSSDKTHTGGGGS
381
E200-'-GS
linker+IgG
hinge+G4S linker
Extended GHNYTTRNILPGLNITSTFHKTSGSGKGGGGSEPKSSDKTHTGG
382
E200+G4S GGS
linker+IgG
hinge+G4S linker
N-term extended DFPGHNYTTRNILPGLNITSEPKSSDKTHT
383
E200 +IgG hinge
N-term extended DFPGHNYTTRNILPGLNITSGSEPKSSDKTHT
384
E200 +GS
linker+IgG hinge
N-term extended DFPGHNYTTRNILPGLNITSGGGGSEPKSSDKTHT
385
E200 +G4S
linker+IgG hinge
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N and C term
DFPGHNYTTRNILPGLNITSTFHKTSGSGKEPKSSDKTHT 386
extended
E200+IgG hinge
N and C term
DFPGHNYTTRNILPGLNITSTFHKTSGSGKGSEPKSSDKTHT 387
extended
E200+GS
linker+IgG hinge
N and C term
DFPGHNYTTRNILPGLNITSTFHKTSGSGKGGGGSEPKSSDKTHT 388
extended E200+
G4S linker+ IgG
hinge
N-term extended DFPGHNYTTRNILPGLNITSEPKSSDKTHTGS
389
E200+IgG hinge
+GS linker
N-term extended DFPGHNYTTRNILPGLNITSGSEPKSSDKTHTGS
390
E200-'-GS
linker+IgG hinge
-'-GS linker
N-term extended DFPGHNYTTRNILPGLNITSGGGGSEPKSSDKTHTGS
391
E200+G4S
linker+IgG hinge
+GS linker
N-term and C
DFPGHNYTTRNILPGLNITSTFHKTSGSGKEPKSSDKTHTGS 392
term extended
E200 + IgG
hinge+GS linker
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N-term and C
DFPGHNYTTRNILPGLNITSTFHKTSGSGKGSEPKSSDKTHTGS 393
term extended
E200 +GS linker+
IgG hinge-'-GS
lin ker
N-term and C
DFPGHNYTTRNILPGLNITSTFHKTSGSGKGGGGSEPKSSDKTHT 394
GS
term extended
E200 + G4S
linker+IgG
hinge+GS linker
N-term extended DFPGHNYTTRNILPGLNITSEPKSSDKTHTGGGGS
395
E200+IgG hinge
+G4S linker
N-term extended DFPGHNYTTRNILPGLNITSGSEPKSSDKTHTGGGGS
396
E200+GS
linker+IgG hinge
+G4S linker
N-term extended DFPGHNYTTRNILPGLNITSGGGGSEPKSSDKTHTGGGGS
397
E200+G4S
linker+IgG hinge
+G4S linker
N-term and C
DFPGHNYTTRNILPGLNITSTFHKTSGSGKEPKSSDKTHTGGGGS 398
term extended
E200 + IgG
hinge+G4S linker
N-term and C
DFPGHNYTTRNILPGLNITSTFHKTSGSGKGSEPKSSDKTHTGGG 399
GS
term extended
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E200 -1-GS linker+
IgG hinge+G4S
lin ker
N-term and C
DFPGHNYTTRNILPGLNITSTFHKTSGSGKGGGGSEPKSSDKTHT 400
term extended .. GGGGS
E200 + G4S
linker+IgG
hinge+G4S linker
Detailed description of the embodiments
[0095] Reference will now be made in detail to certain embodiments of the
invention.
While the invention will be described in conjunction with the embodiments, it
will be
understood that the intention is not to limit the invention to those
embodiments. On the
contrary, the invention is intended to cover all alternatives, modifications,
and
equivalents, which may be included within the scope of the present invention
as defined
by the claims.
[0096] One skilled in the art will recognise many methods and materials
similar or
equivalent to those described herein, which could be used in the practice of
the present
invention. The present invention is in no way limited to the methods and
materials
described.
[0097] It will be understood that the invention disclosed and defined in this
specification extends to all alternative combinations of two or more of the
individual
features mentioned or evident from the text or drawings. All of these
different
combinations constitute various alternative aspects of the invention.
[0098] All of the patents and publications referred to herein are incorporated
by
reference in their entirety.
[0099] The present invention seeks to address one or more of the deficiencies
of the
prior art and is based on the recognition by the inventors that it is possible
to exploit the
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cancer-specific expression of tumour-specific antigens, such as a
dysfunctional P2X7
receptor, to:
a) expand the targets of immune cells expressing chimeric antigen receptors
(CARs) which bind to a tumour-specific antigen, such as a dysfunctional P2X7
receptor; and/or
b) provide a tunable, "switchable" approach to targeted cell killing in a
variety of
settings that minimise on-target, off-tumour effects; and
c) minimise aberrant immune responses to adaptor molecules.
[0100] The invention provides a new treatment modality comprising a first
component
and second component. The first component is the administration of an immune
cell
that comprises a CAR that binds to a dysfunctional P2X7 receptor expressed on
a cell
surface (also referred to herein as "nfP2X7-directed CAR"). The second
component is
the administration (or expression) of a bridging molecule that can redirect
tumour-
specific CARs (eg nfP2X7-directed CARs) to other cell surface molecules, such
as
cancer-associated antigens, e.g. CD19, CD20, CD33, GD2, intracellularly
processed
proteins that are presented as peptides of various length via MHC I and ll or
via any
other mechanism of accessible surface antigen exposure.
[0101] The targeted cell surface molecules may be associated with cancer (such
as
tumour associated antigens expressed on the surface of cancer cells). In
further
embodiments, the targeted cell surface molecules may be associated with an
infection,
or associated with any other disease (including autoimmune diseases). In such
embodiments, the molecules may be cell surface antigens associated with the
disease
or may include peptide/HLA complexes presented on cells. In certain examples
the
molecule may comprise CD19 in B-lineage malignancy. The molecule may comprise
targeted peptides related to cancer-specific proteins (genetic aberrations
such as
cancer testis antigens and others which are specific to the cancer patient).
The
molecule may be a peptide/HLA complex comprising peptides derived from an
infectious agent e.g. of viral, bacterial, protozoan, virion, prion and fungal
origin. The
molecule may be a peptide/HLA complex comprising peptides associated with an
autoimmune disease (e.g., Sm peptides associated with lupus).
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[0102] Further, the present invention provides several advantages over
existing
"adaptor CAR" approaches. In more detail, adaptor CAR T cell technology is
based on
an approach to uncoupling the tumour-targeting and signalling moieties of
conventional
CARs, resulting in dichotomous systems consisting of an adaptor CAR and
soluble,
tumour-specific adaptor molecules. The basic structure of adaptor CARs
corresponds to
the conventional CAR design, although the extracellular domain does not
interact with
the tumour-associated antigen, but instead with a binding partner incorporated
into the
adaptor molecule. The bifunctional adaptor molecule in turn provides target
cell (tumour
cell) specificity and acts as a linker at the interface between the tumour and
the adaptor
CAR T cell. This complex can then mediate anti-tumour responses, similarly to
conventional CAR T cells.
[0103] While the dual principle of the adaptor CAR systems provides an
important
molecular safety switch to precisely control the adaptor CAR T cell activity,
several
limitations have been identified with the systems developed to date.
[0104] For example, one system relies on scFv-based a-FITC CARs targeting the
synthetic dye FITC that is coupled to various tumour-specific adaptor
molecules.
Despite the full humanisation of the a-FITC CAR T cell product, the
immunogenic
potential of FITC is one concern for clinical translation, as underlined by
the emergence
of anti-a-FITC antibodies in therapeutic mouse models.
[0105] In another example, adaptor molecules include small peptide tags to
redirect
standard scFv-based adaptor CARs. However, concerns again have been raised
with
respect to the immunogenic potential of the small peptide tags included in the
adaptor
molecules, particularly in the case where the tag comprises non-human
sequences, or
sequences derived from human nuclear proteins.
[0106] In contrast to the approaches of the prior art, the present invention
takes
advantage of both the specificity of CARs that bind dysfunctional P2X7 and of
the
unique properties of epitopes derived from dysfunctional P2X7 receptor.
[0107] The present invention provides an advantage over existing mono-antigen
directed CAR T therapy in that it:
= makes use of CAR T cells that are functional (i.e., there is no
requirement for the
use of a switching molecule to activate the CAR T cells);
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= utilises CAR T cells that do not bind to healthy cells since
dysfunctional P2X7
receptor is only exposed on the surface of cancer cells;
o this means that there is no requirement for the administration of
immunosuppressing agents;
o this also means that there is a significant reduction in the side-effects
as
compared to other approaches that include the use of CARs that bind
antigens also found on non-cancerous cells;
= comprises non-immunogenic, and naturally occurring human epitopes on the
bridging molecule in the form of epitopes derived from dysfunctional P2X7
receptor.
[0108] Thus the present invention provides a new concept and approach in the
use of
adaptor/bridging molecules alongside CAR T therapy.
[0109] In more detail, the specificity of CARs that bind dysfunctional nfP2X7
receptor
(also referred to herein as nfP2X7 CAR) results from the fact that
dysfunctional P2X7 is
only exposed on the surface of transformed cells. Further, by including an
epitope from
nfP2X7 in a bridging molecule, CAR-mediated recognition can be broadened to
include
any target antigen of interest via the corresponding bridging molecule. The
nfP2X7 CAR
solely recognises the dysfunctional P2X7 receptor, e.g. the E200 (or E300 or
E200-300
composite) epitope, however the use of a bridging molecule facilitates
unlimited
targeting by means of any accessible recognition site expressed on the cell
surface, e.g.
an nfP2X7 CAR can be additionally directed (or redirected) to bind to CD19
positive
cells through the use of a bridging molecule that comprises a targeting moiety
for
binding CD19 on a cell surface, and an E200 epitope from nfP2X7.
[0110] P2X7 is a human receptor protein that is commonly expressed in human
tissue, particularly immune and neural cells. There is no reported or
registered case of
autoimmune response raised against P2X7 receptors. Exemplary targeted epitopes

such as E200, E300 and E200/E300 are not genetically defined but only result
from a
conformational change of the tertiary structure of P2X7. Thus, these are non-
immunogenic peptide sequences that are an unaltered part of the P2X7 sequence.
Only
under artificial conditions using adjuvants and conjugates can immune
responses be
produced against the target.
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[0111] An advantage of the non-immunogenic recognition sites, e.g. the peptide

sequence of E200 or E300 or the composite peptide E200-300, in the bridging
molecule
facilitates long-term application of bridging molecules with various
specificities without
induction of neutralising antibodies and/or T cell mediated rejection of
cells. This
represents a significant advantage over the design of bridging/adaptor
molecules
described in the prior art.
[0112] Moreover, nfP2X7 CARs only target cancer tissue specifically, and
therefore
the approach of the present invention presents minimal risk of "on-target, off-
tumour"
effects and damage to healthy tissue through off-target binding of the CARs.
The
present invention therefore exploits the specificity of nfP2X7 CARs in two
ways: firstly by
relying on the fact that nfP2X7 CARs only target nfP2X7 expressing cells
(cancer cells
only) and secondly, by relying on the fact that bridging molecules, engineered
to be
bound by nfP2X7 can be used to redirect immune cells expressing nfP2X7 CARs
towards other tumour-associated and/or specific target antigens in a
switchable,
tuneable manner.
[0113] Thus, the use of the bridging molecules of the invention allows for the

redirection of immune cells expressing nfP2X7 CARs for the targeting of any
surface
expressed target antigen.
[0114] A particular advantage of the present approach is that the targeting is
limited
to the time period during which the bridging molecule therapeutic in vivo is
persistent in
circulation. This means that any toxicity arising from the "on-target, off-
tumour"
expression of the target antigen in healthy tissue is minimised. This is
because once the
bridging molecule has been cleared from the body, nfP2X7 CAR cells are again
only
capable of tumour-specifically targeting nfP2X7. In other words, as the
targeting of
target antigens other than nfP2X7 is bridging molecule-dependent, the
targeting can be
regulated by the application of bridging molecules. This facilitates an easy
to implement
approach for "switching-on" and "switching-off' the targeting of cancer cells
via the
antigen bound by the targeting moiety of the bridging molecules such that the
CAR T
cells may be transiently directed to cancer cells via antigens other than
nfP2X7 Further,
the length of time during which the CAR T cells are redirected to other cancer
antigens
can be modulated by the time for which the bridging molecules are administered
to a
patient in need.
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[0115] It should therefore be clear that the present invention finds
application in a
variety of settings. For example, in the context of oncology treatment, the
present
invention allows for the use of a single class of CAR molecule (i.e., for
binding
dysfunctional P2X7 receptor present on cancer cells), to target multiple
antigens present
on the cancer cells. More specifically, using a bridging molecule that
comprises a
targeting moiety for binding CD19, the CAR-T cells can be targeted to the
cancer cells
at both dysfunctional P2X7 receptor and CD19. This maximises the likelihood of
the
cancer cell being killed because it is being targeted at multiple sites.
Moreover, it should
be evident that the use of multiple bridging molecules, or bridging molecules
comprising
more than one targeting moiety, facilitates the "painting" of the cancer cell
surface with
CAR T cells. In other words, the invention provides for the use of a variety
of different
bridging molecules, each comprising epitopes for being bound by a single
species of
CAR T cell, but comprising different targeting moieties such that the CAR T
cells may
be directed to bind to cancer cells via multiple cancer antigens. In this way,
the cancer
cells can be targeted and bound by the CAR T cell at multiple sites,
increasing the
efficacy of cell killing.
[0116] This approach is also particularly useful in the case of cancers that
express
low levels of dysfunctional P2X7 receptor, such as Burkitt's lymphoma or
subcategories
of solid tumours arising from various epithelial, mesenchymal, neural or
germinal
origins. Another example of a low-expressing cancer cell type may be the
triple negative
breast cancer (e.g. MDA-MB-231 cell line). Other examples include solid tumour
tissues
tested in tissue arrays from PDX models, several of which show lower receptor
expression than other cancers. Such examples include but are not limited to
neuroblastoma, colorectal cancer, lung cancer, breast cancer or brain cancer.
[0117] In further examples, the invention finds application in the context of
preventing
or minimising the severity of an infection with a pathogen (preferably an
intracellular
pathogen). While not limited to an oncology setting, this may be particularly
useful in the
treatment of patients receiving cancer therapy and who are immunocompromised
(and
therefore susceptible to infection with opportunistic or other pathogens).
Thus, a patient
who has received (or is continuing to receive) a treatment with CAR T cells
that bind
dysfunctional P2X7 receptor, can simultaneously be administered a bridging
molecule
that facilitates the redirection of the CAR T cells to cells that present
peptides from an
infectious agent on MHC molecules on their cell surface. In other words, the
invention
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provides a platform for simultaneous or sequential treatment of cancer and an
infectious
disease.
[0118] The basic principle as well as the engagement of nfPX7 CAR expressing
effector cells via nfP2X7 E200-derived peptide tagged bridging molecules and
the
different formats of bridging molecules, is illustrated and outlined in
Figures 1 to 3.
[0119] Using nfP2X7R CAR without the presence of bridging molecules, the
nfP2X7
CAR expressing effector cells exhibit cancer-specific targeting (scenario I,
see Figure
11).
[0120] In order to broaden the applicability to nfP2X7 functionally negative
cancers
(very low or negative for nfP2X7) nfP2X7 CAR expressing effector cells may be
redirected to cancer cells via bridging molecules targeting cancer-associated
antigens
as illustrated for CD33 or cancer-specific antigens via TcR-like mAbs. The
specificity of
the bridging molecules is unlimited meaning any surface expressed target
antigen or
presented antigen in the context of MHC peptide presentation (class I and II)
via TcR-
like mAb or ligands may engage the nfP2X7 CAR expressing effector cells in the
same
mode of action (scenario II. See Figure 11).
[0121] In most cases, the dual-function of the nfP2X7 CAR expressing effector
cells is
utilised (scenario III, see Figure 11). It is a combination of scenario I. and
II, which
means that nfP2X7 CAR expressing effector cells are engaged directly to cancer
cells
via nfP2X7 expressed on the cancer cells and additionally recruited to the
cancer cells
via bridging molecules targeting cancer-associated antigens as illustrated for
CD33 or
cancer-specific antigens via TcR-like mAbs.
Definitions
[0122] Unless defined otherwise, technical and scientific terms used herein
have the
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs.
[0123]
For purposes of interpreting this specification, the following definitions
will
generally apply and whenever appropriate, terms used in the singular will also
include
the plural and vice versa.
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[0124] "Purinergic receptor" generally refers to a receptor that uses a purine
(such as
ATP) as a ligand.
[0125] "P2X7 receptor" generally refers to a purinergic receptor formed from
three
protein subunits or monomers, with at least one of the monomers having an
amino acid
sequence substantially as shown in SEQ ID NO: 1 below:
[0126] SEQ ID NO: 1
M PACCSCSDVFQYETNKVTRIQSMNYGTIKWFFHVI IFSYVCFALVSDKLYQRKEPVIS
SVHTKVKG IAEVKE E IVENGVKKLVHSVFDTADYTFP LOG NSFFVMTN FLKTEGQEQRL
CPEYPTRRTLCSSDRGCKKGWMDPOSKGIQTGRCVVYEGNQKTCEVSAWCPIEAVE
EAPRPALLNSAENFTVL IKNNI DF PG H NYTTRN ILPGLNITCTFHKTQNPQCPIFRLGDIF
RETGDNFSDVAIQGGIMGIEIYWDCNLDRWFHHCRPKYSFRRLDDKTTNVSLYPGYNF
RYAKYYKENNVEKRTLIKVFG IRFDILVFGTGGKFDIIQLVVYIGSTLSYFGLAAVFIDFLID
TYSSNCCRSH IYPWCKCCQPCVVN EYYYRKKCESIVEPKPTLKYVSFVDESH IRMVNQ
QLLGRSLQDVKGQEVPRPAMDFTDLSRLPLALHDTPPIPGQPEEIQLLRKEATPRSRD
SPVWCQCGSCLPSQLPESHRCLEELCCRKKPGACITTSELFRKLVLSRHVLQFLLLYQ
EPLLALDVDSTNSRLRHCAYRCYATWRFGSQDMADFAILPSCCRWRIRKEFPKSEGQ
YSGFKSPY
[0127] To the extent that P2X7 receptor is formed from three monomers, it is a

"trimer" or "trimeric". "P2X7 receptor" encompasses naturally occurring
variants of P2X7
receptor, e.g., wherein the P2X7 monomers are splice variants, allelic
variants, SNPs
and isoforms including naturally-occurring truncated or secreted forms of the
monomers
forming the P2X7 receptor (e.g., a form consisting of the extracellular domain
sequence
or truncated form of it), naturally-occurring variant forms (e.g.,
alternatively spliced
forms) and naturally-occurring allelic variants. In certain embodiments of the
invention,
the native sequence P2X7 monomeric polypeptides disclosed herein are mature or
full-
length native sequence polypeptides comprising the full-length amino acids
sequence
shown in SEQ ID NO: 1. In certain embodiments the P2X7 receptor may have an
amino
acid sequence that is modified, for example various of the amino acids in the
sequence
shown in SEQ ID NO: 1 may be substituted, deleted, or a residue may be
inserted.
[0128] "Functional P2X7 receptor" generally refers to a form of the P2X7
receptor
having three intact binding sites or clefts for binding to ATP. When bound to
ATP, the
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functional receptor forms a non-selective sodium/calcium channel that converts
to a
pore-like structure that enables the ingress of calcium ions and molecules of
up to 900
Da into the cytosol, one consequence of which may be induction of programmed
cell
death. In normal homeostasis, expression of functional P2X7 receptors is
generally
limited to cells that undergo programmed cell death such as thymocytes,
dendritic cells,
lymphocytes, macrophages and monocytes. There may also be some expression of
functional P2X7 receptors on erythrocytes and other cell types.
[0129] "Dysfunctional P2X7 receptor" generally refers to a form of a P2X7
receptor
having a conformation, distinct from functional P2X7, whereby the receptor is
unable to
form an apoptotic pore, but which is still able to operate as a non-selective
channel
through the maintenance of a single functional ATP binding site located
between
adjacent monomers. One example arises where one or more of the monomers has a
cis
isomerisation at Pro210 (according to SEQ ID NO: 1). The isomerisation may
arise from
any molecular event that leads to misfolding of the monomer, including for
example,
mutation of monomer primary sequence or abnormal post translational
processing. One
consequence of the isomerisation is that the receptor is unable to bind to ATP
at one, or
more particularly two, ATP binding sites on the trimer and as a consequence
not be
able to extend the opening of the channel. In the circumstances, the receptor
cannot
form a pore and this limits the extent to which calcium ions may enter the
cytosol.
Dysfunctional P2X7 receptors are expressed on a wide range of epithelial and
haematopoietic cancers. As used herein, the term "dysfunctional P2X7
receptors" may
be used interchangeably with the term "non-functional P2X7 receptors" or
"nfP2X7
receptors".
[0130] "Cancer associated-P2X7 receptors" are generally P2X7 receptors that
are
found on cancer cells (including, pre-neoplastic, neoplastic, malignant,
benign or
metastatic cells), but not on non-cancer or normal cells.
[0131] "E200 epitope" generally refers to an epitope having the sequence
GHNYTTNILPGLNITC and variants thereof (e.g. SEQ ID NOs: 2-11, 15-30 and 168).
[0132] "E300 epitope" generally refers to an epitope having the sequence
KYYKENNVEKRTLIK and variants thereof (SEQ ID NOs: 12 and 13).
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[0133] A "composite epitope" generally refers to an epitope that is formed
from the
juxtaposition of the E200 and E300 epitopes or parts of these epitopes. An
example of a
composite epitope comprising E200 and E300
epitopes is
GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 14).
[0134] "Antibodies" or "immunoglobulins" or "Igs" are gamma globulin proteins
that
are found in blood, or other bodily fluids of vertebrates that function in the
immune
system to bind antigen, hence identifying and/or neutralising foreign objects.
[0135] Antibodies are generally a heterotetrameric glycoprotein composed of
two
identical light (L) chains and two identical heavy (H) chains. Each L chain is
linked to a
H chain by one covalent disulfide bond. The two H chains are linked to each
other by
one or more disulfide bonds depending on the H chain isotype. Each H and L
chain also
has regularly spaced intrachain disulfide bridges.
[0136] H and L chains define specific Ig domains. More particularly, each H
chain has
at the N-terminus, a variable domain (VH) followed by three constant domains
(CH) for
each of the a and y chains and four CH domains for p and c isotypes. Each L
chain has
at the N-terminus, a variable domain (VL) followed by a constant domain (CL)
at its
other end. The VL is aligned with the VH and the CL is aligned with the first
constant
domain of the heavy chain (CHI).
[0137] Antibodies can be assigned to different classes or isotypes. There are
five
classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains
designated a, 6, , y, and p, respectively. The y and a classes are further
divided into
subclasses on the basis of relatively minor differences in 3/4 sequence and
function, e.g.,
humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgAI, and
IgA2. The
L chain from any vertebrate species can be assigned to one of two clearly
distinct types,
called kappa and lambda, based on the amino acid sequences of their constant
domains.
[0138] The constant domain includes the Fc portion that comprises the carboxy-
terminal portions of both H chains held together by disulfides. The effector
functions of
antibodies such as ADCC are determined by sequences in the Fc region, which
region
is also the part recognised by Fc receptors (FcR) found on certain types of
cells.
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[0139] The pairing of a VH and VL together forms a "variable region" or
"variable
domain" including the amino -terminal domains of the heavy or light chain of
the
antibody. The variable domain of the heavy chain may be referred to as "VH."
The
variable domain of the light chain may be referred to as "VL." The V domain
contains an
"antigen binding site" that affects antigen binding and defines specificity of
a particular
antibody for its particular antigen. V regions span about 110 amino acid
residues and
consist of relatively invariant stretches called framework regions (FRs)
(generally about
4) of 15-30 amino acids separated by shorter regions of extreme variability
called
"hypervariable regions" (generally about 3) that are each generally 9-12 amino
acids
long. The FRs largely adopt a p-sheet configuration and the hypervariable
regions form
loops connecting, and in some cases forming part of, the p-sheet structure.
[0140] "Hypervariable region" refers to the regions of an antibody variable
domain
that are hypervariable in sequence and/or form structurally defined loops.
Generally,
antibodies comprise six hypervariable regions; three in the VH (H1, H2, H3),
and three in
the VL (L1, L2, L3).
[0141] "Framework" or "FR" residues are those variable domain residues other
than
the hypervariable region residues herein defined.
[0142] An "antigen binding site" generally refers to a molecule that includes
at least
the hypervariable and framework regions that are required for imparting
antigen binding
function to a V domain. An antigen binding site may be in the form of an
antibody or an
antibody fragment, (such as a mAb, single domain (SD)-mAb, dAb, Fab, SD-Fab,
Fd,
SD-Fv, Fv, F(ab')2 or scFv) in a method described herein.
[0143] An "intact" or "whole" antibody is one that comprises an antigen-
binding site as
well as a CL and at least heavy chain constant domains, CH1, CH2 and CH3. The
constant domains may be native sequence constant domains (e.g human native
sequence constant domains) or amino acid sequence variant thereof.
[0144] "Whole antibody fragments including a variable domain" include SD-mAb,
Fab,
Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies, single-chain
antibody
molecules; and multi-specific antibodies formed from antibody fragments.
[0145] The "Fab fragment" consists of an entire L chain along with the
variable region
domain of the H chain (VH), and the first constant domain of one heavy chain
(CH1).
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Each Fab fragment is monovalent with respect to antigen binding, i.e., it has
a single
antigen-binding site.
[0146] A ''Fab fragment" differs from Fab fragments by having additional few
residues
at the carboxy terminus of the CH1 domain including one or more cysteines from
the
antibody hinge region. Fab'- SH is the designation herein for Fab' in which
the cysteine
residue(s) of the constant domains bear a free thiol group.
[0147] A "F(ab')2 fragment" roughly corresponds to two disulphide linked Fab
fragments having divalent antigen-binding activity and is still capable of
cross-linking
antigen.
[0148] An "Fv" is the minimum antibody fragment that contains a complete
antigen-
recognition and binding site. This fragment consists of a dimer of one heavy
and one
light chain variable region domain in tight, non-covalent association.
[0149] In a single-chain Fv (scFv) species, one heavy and one light chain
variable
domain can be covalently linked by a flexible peptide linker such that the
light and heavy
chains can associate in a "dimeric" structure analogous to that in a two-chain
Fv
species. From the folding of these two domains emanate six hypervariable loops
(3
loops each from the H and L chain) that contribute the amino acid residues for
antigen
binding and confer antigen binding specificity to the antibody.
[0150] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody
fragments
that comprise the VH and VL antibody domains connected to form a single
polypeptide
chain. Preferably, the scFv polypeptide further comprises a polypeptide linker
between
the VH and VL domains that enables the scFv to form the desired structure for
antigen
binding.
[0151] A "single variable domain" is half of an Fv (comprising only three CDRs

specific for an antigen) that has the ability to recognise and bind antigen,
although
generally at a lower affinity than the entire binding site.
[0152] "Diabodies" refers to antibody fragments with two antigen-binding
sites, which
fragments comprise a heavy-chain variable domain (VH) connected to a light-
chain
variable domain (VL) in the same polypeptide chain (VH-VL). The small antibody

fragments are prepared by constructing sFy fragments (see preceding paragraph)
with
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short linkers (about 5-10 residues) between the VH and VL domains such that
interchain
but not intra-chain pairing of the V domains is achieved, resulting in a
bivalent fragment,
i.e., a fragment having two antigen-binding sites.
[0153] Diabodies may be bivalent or bispecific. Bispecific diabodies are
heterodimers
of two "crossover" sFy fragments in which the VH and VL domains of the two
antibodies
are present on different polypeptide chains. Triabodies and tetrabodies are
also
generally known in the art.
[0154] An "isolated antibody" is one that has been identified and separated
and/or
recovered from a component of its pre-existing environment. Contaminant
components
are materials that would interfere with therapeutic uses for the antibody, and
may
include enzymes, hormones, and other proteinaceous or non-proteinaceous
solutes.
[0155] A "human antibody" refers to an antibody that possesses an amino acid
sequence that corresponds to that of an antibody produced by a human. Human
antibodies can be produced using various techniques known in the art,
including phage
-display libraries. Human antibodies can be prepared by administering the
antigen to a
transgenic animal that has been modified to produce such antibodies in
response to
antigenic challenge, but whose endogenous loci have been disabled.
[0156] "Humanised' forms of non-human (e.g., rodent) antibodies are chimeric
antibodies that contain minimal sequence derived from the non-human antibody.
For the
most part, humanised antibodies are human immunoglobulins (recipient antibody)
in
which residues from a hypervariable region of the recipient are replaced by
residues
from a hypervariable region of a non-human species (donor antibody) such as
mouse,
rat, rabbit or non-human primate having the desired antibody specificity,
affinity, and
capability. In some instances, framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human residues. Furthermore,
humanised antibodies may comprise residues that are not found in the recipient

antibody or in the donor antibody. These modifications are made to further
refine
antibody performance. In general, the humanised antibody will comprise
substantially all
of at least one, and typically two, variable domains, in which all or
substantially all of the
hypervariable loops correspond to those of a non-human immunoglobulin and all
or
substantially all of the FRs are those of a human immunoglobulin sequence. The
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humanised antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
[0157] "Monoclonal antibody" refers to an antibody obtained from a population
of
substantially homogeneous antibodies, i.e., the individual antibodies
comprising the
population are identical except for possible naturally occurring mutations
that may be
present in minor amounts. Monoclonal antibodies are highly specific, being
directed
against a single antigenic site or determinant on the antigen. In addition to
their
specificity, the monoclonal antibodies are advantageous in that they may be
synthesised uncontaminated by other antibodies. Monoclonal antibodies may be
prepared by the hybridoma methodology. The "monoclonal antibodies" may also be

isolated from phage antibody libraries using molecular engineering techniques.
[0158] The term "anti-P2X7 receptor antibody" or "an antibody that binds to
P2X7
receptor" refers to an antibody that is capable of binding P2X7 receptor with
sufficient
affinity such that the antibody is useful as a diagnostic and/or therapeutic
agent in
targeting P2X7 receptor, typically non-functional P2X7 receptor or a cancer
associated
P2X7 receptor. Preferably, the extent of binding of a P2X7 receptor antibody
to an
unrelated protein is less than about 10% of the binding of the antibody to
P2X7 receptor
as measured, e.g., by a radioimmunoassay (RIA), Enzyme-Linked Immunosorbent
Assay (ELISA), Biacore or Flow Cytometry. In certain embodiments, an antibody
that
binds to P2X7 receptor has a dissociation constant (Kd) of < 1 pM, < 100 nM, <
10 nM,
< 1 nM, or < 0.1 nM. An anti nfP2X7 receptor antibody is generally one having
some or
all of these serological characteristics and that binds to dysfunctional P2X7
receptors
but not to functional P2X7 receptors.
[0159] An "affinity matured' antibody is one with one or more alterations in
one or
more hypervariable regions thereof that result in an improvement in the
affinity of the
antibody for the antigen, compared to a parent antibody that does not possess
those
alteration(s). Preferred affinity matured antibodies will have nanomolar or
even
picomolar affinities for the target antigen. Affinity matured antibodies are
produced by
procedures known in the art.
[0160] A "blocking" antibody" or an "antagonist" antibody is one that inhibits
or
reduces biological activity of the antigen it binds. Preferred blocking
antibodies or
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antagonist antibodies substantially or completely inhibit the biological
activity of the
antigen.
[0161] An "agonist antibody", as used herein, is an antibody, which mimics at
least
one of the functional activities of a polypeptide of interest.
[0162] "Binding affinity" generally refers to the strength of the sum total of
non-
covalent interactions between a single binding site of a molecule (e.g., an
antibody) and
its binding partner (e.g., an antigen). Unless indicated otherwise, as used
herein,
"binding affinity" refers to intrinsic binding affinity, which reflects a 1 :
1 interaction
between members of a binding pair (e.g., antibody and antigen). The affinity
of a
molecule X for its partner Y can generally be represented by the dissociation
constant
(Kd). Affinity can be measured by common methods known in the art, including
those
described herein. Low-affinity antibodies generally bind antigen slowly and
tend to
dissociate readily, whereas high-affinity antibodies generally bind antigen
faster and
tend to remain bound longer. A variety of methods of measuring binding
affinity are
known in the art, any of which can be used for purposes of the present
invention.
[0163] As used herein, the term "antigen" is intended to include substances
that bind
to or evoke the production of one or more antibodies and may comprise, but is
not
limited to, proteins, peptides, polypeptides, oligopeptides, lipids,
carbohydrates, and
combinations thereof, for example a glycosylated protein or a glycolipid. The
term
"antigen" as used herein refers to a molecular entity that may be expressed on
a target
cell and that can be recognised by means of the adaptive immune system
including but
not restricted to antibodies or TCRs, or engineered molecules including but
not
restricted to transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments
or
multimers thereof, antibodies or multimers thereof, single chain antibodies or
multimers
thereof, or any other molecule that can execute binding to a structure with
high affinity.
[0164] "Epitope" generally refers to that part of an antigen that is bound by
the
antigen binding site of an antibody. An epitope may be "linear" in the sense
that the
hypervariable loops of the antibody CDRs that form the antigen binding site
bind to a
sequence of amino acids as in a primary protein structure. In certain
embodiments, the
epitope is a "conformational epitope" i.e. one in which the hypervariable
loops of the
CDRs bind to residues as they are presented in the tertiary or quaternary
protein
structure.
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[0165] The term "target cell" as used herein refers to a cell that expresses a

dysfunctional P2X7 receptor (e.g. nfP2X7 receptor) or a cell surface molecule
to which
the targeting moiety of the bridging molecule binds. The target cell may be a
cancer cell
or any other diseased cell.
[0166] The term "disorder" or "condition" means a functional abnormality or
disturbance in a subject such as a cancer, an autoimmune disorder, or an
infection by
virus, bacteria, parasite, or others.
[0167] For example, a nucleic acid or a peptide naturally present in a living
animal is
not "isolated", but the same nucleic acid or peptide partially or completely
separated
from the coexisting materials of its natural state is "isolated". An isolated
nucleic acid or
protein can also exist in a non-native environment such as, for example, in a
host cell.
[0168] As used herein, the term "subject" refers to a mammal such as mouse,
rat,
cow, pig, goat, chicken, dog, monkey or human. Preferentially, the subject is
a human.
The subject may be a subject suffering from a disorder such as cancer (a
patient), but
the subject also may be a healthy subject.
[0169] The term "autologous" as used herein refers to any material derived
from the
same subject to whom it is later re-introduced.
[0170] The term "allogeneic" as used herein refers to any material derived
from a
different subject of the same species as the subject to whom the material is
re-
introduced.
[0171] The terms "therapeutically effective amount" or "therapeutically
effective
population" mean an amount of, for example, a cell population that provides a
therapeutic benefit in a subject.
[0172] The terms "binds to", "specifically binds to" or "specific for" with
respect to a
targeting moiety, as used e.g. in the bridging molecule as disclosed herein,
or of a CAR
referring to an antigen-binding domain that recognises and binds to a specific
antigen,
does not substantially recognise or bind to other molecules in a sample. An
antigen-
binding domain or targeting moiety that binds specifically to an antigen from
one
species also may bind to that antigen from another species. This cross-species

reactivity is typical of many antibodies and therefore not contrary to the
definition that
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the antigen-binding domain is specific. An antigen-binding domain that
specifically binds
to an antigen may bind also to different allelic forms of the antigen (allelic
variants,
splice variants, isoforms etc.) or homologous variants of this antigen from
the same
gene family. This cross reactivity is typical of many antibodies and therefore
not
contrary to the definition that the antigen-binding domain is specific.
[0173] The terms "engineered cell" and "genetically modified cell" as used
herein can
be used interchangeably. The terms mean containing and/or expressing a foreign
gene
or nucleic acid sequence that in turn modifies the genotype or phenotype of
the cell or
its progeny. Especially, the terms refer to the fact that cells,
preferentially immune cells,
can be manipulated by recombinant methods well known in the art to express
stably or
transiently peptides or proteins that are not expressed in these cells in the
natural state.
For example, immune cells are engineered to express an artificial construct
such as a
chimeric antigen receptor on their cell surface. For example, the CAR
sequences may
be delivered into cells using an adenoviral, adeno-associated viral (AAV)-
based,
retroviral or lentiviral vector or any other pseudotyped variations thereof or
any other
gene delivery mechanism such as electroporation or lipofection with
CRISPR/Cas9,
transposons (e.g. sleeping-beauty) or variations thereof. The gene delivery
may be in
the form of mRNA (transient) or DNA (transient or permanent).
[0174] The terms "immune cell" or "immune effector cell" refer to a cell that
may be
part of the immune system and executes a particular effector function such as
alpha-
beta T cells, NK cells, NKT cells, B cells, Breg cells, Treg cells, innate
lymphoid cells
(ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK)
cells,
gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells
(MSC),
monocytes or macrophages or any hematopoietic progenitor cells such as
pluripotent
stem cells and early progenitor subsets that may mature or differentiate into
somatic
cells. The cells may be naturally occurring or generated by cytokine exposure,

artificial/genetically modified cells (such as iPSCs and other artificial cell
types). The
immune cell may be an artificial cell subset including induced pluripotent
stem cells and
cells maturated therefrom. Preferred immune cells are cells with cytotoxic
effector
function such as alpha-beta T cells, NK cells, NKT cells, ILC, CIK cells, LAK
cells or
gamma-delta T cells. "Effector function" means a specialised function of a
cell, e.g. in a
T cell an effector function may be cytolytic activity or helper cell activity
including the
secretion of cytokines.
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[0175] The term "treat" (treatment of) a disorder as used herein means to
reduce the
frequency or severity of at least one sign or symptom of a disease or disorder

experienced by a subject.
[0176] The term "expression" as used herein is defined as the transcription
and/or
translation of a particular nucleotide sequence driven by its promoter in a
cell.
Chimeric antigen receptor (CAR)
[0177] The term cancer-specific CAR (T cell) targeting refers to the use of a
CAR T
cell for binding to a target antigen that is presented on the cell surface of
tumour cells,
but is not typically found on the surface of a healthy cell. In other words,
normal cells
under normal circumstances may be characterised by the absence of the target
antigen
on the extracellular membrane (and therefore the presence of the antigen on
the cell
surface cannot be detected). However, such cells may express mRNA encoding the

antigen at an intracellular level. As CAR T cells only recognize surface-
expressed
antigens, the intracellular expression of the targeted proteins will not lead
to CAR
engagement.
[0178] The targeted epitopes E200 and E300 of the P2X7 receptor are not
exposed
on the form of the receptor found in healthy tissue and thus these epitopes
can be
regarded as cancer specific. In other words, the E200 and E300 epitopes are
only
exposed, and available for binding when the P2X7 receptor has an altered non-
functional conformation, such as occurs in the context of cancer (in which
case the
receptor is referred to as nfP2X7 receptor). Another example of a cancer-
specific
targeted epitope may be derived from the splice variant EGFRvIll. Still
another example
is the antigen CLDN6 which is mostly restricted to embryonic and foetal life
and has
very limited expression in healthy cells after the early phase in life and may
be regarded
as highly restricted and relatively overexpressed in cancer. The present
invention
contemplates the binding any such tumour-specific antigen, including nfP2X7,
EGFRvIll
and CLDN6 for cancer-specific targeting and engaging CAR T cells via the
bridging
molecules described herein to cancer-associated antigens.
[0179] In general, a CAR may comprise an extracellular domain (extracellular
part)
comprising the antigen binding domain, a transmembrane domain and an
intracellular
signaling domain. The extracellular domain may be linked to the transmembrane
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domain by a linker. The extracellular domain may also comprise a signal
peptide. The
extracellular part of the CAR of the present invention comprises a tumour-
specific
antigen binding domain. For example, the tumour-specific antigen may be any
one
described herein, including nfP2X7, EGFRvIll or CLDN6.
[0180] The tumour-specific antigen binding domain may be a nfP2X7 binding
domain
that recognises the E200 (or E300 or E200-300 composite) epitope as disclosed
herein.
Specifically, the CAR as disclosed herein has an extracellular nfP2X7 E200 (or
E300 or
E200-300 composite) binding domain as an antigen binding domain.
Alternatively, the
tumour-specific antigen binding domain may be an EGFRvIll binding domain that
recognises an epitope resulting out of the fusion of the amino acid sequence
starting at
position 25-29 LEEKK, followed by the insertion of G and the subsequent amino
acid
sequence 298-304 NYVVTDH, the total epitope is a 13-mer comprised of the
sequence
LEEKKGNYVVTDH (SEQ ID NO: 267). Alternatively, the tumour-specific antigen
binding domain may be a CLDN6 binding domain that recognises an epitope in the

second extracellular domain of CLDN6 [UniProtKB - P56747 (CLDN6_HUMAN)] via
the
amino acid sequence of SEQ ID NO: 273, 274 or 275.
[0181] Typically, the antigen-recognition domain includes a binding
polypeptide that
includes amino acid sequence homology to one or more complennentarity
determining
regions (CDRs) of an antibody that binds to a tumour-specific antigen (such as
a
dysfunctional P2X7 receptor, EGFRvIll or CLDN6). In any embodiment, the
binding
polypeptide includes amino acid sequence homology to the CDR1, 2 and 3 domains
of
the VH and/or VL chain of an antibody that binds to a tumour-specific antigen
(such as a
dysfunctional P2X7 receptor, EGFRvIll or CLDN6).
[0182] In particularly preferred embodiments of the invention, the antigen-
recognition
domain of the CAR binds to an epitope of the tumour-specific antigen nfP2X7
[0183] In such embodiments, the binding polypeptide comprises the amino acid
sequence of the CDRs of the VH and/or VL chain of an antibody described in any
one of:
PCT/AU2002/000061 or PCT/AU2002/001204 (or in any one of the corresponding US
patents US 7,326,415, US 7,888,473, US 7,531,171, US 8,080,635, US 8,399,617,
US
8,709,425, US 9,663,584, or US 10,450,380), PCT/AU2007/001540 (or in
corresponding US patent US 8,067,550), PCT/AU2007/001541 (or in corresponding
US
publication US 2010-0036101), PCT/AU2008/001364 (or in any one of the
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corresponding US patents US 8,440,186, US 9,181,320, US 9,944,701 or US
10,597,451), PCT/AU2008/001365 (or in any one of the corresponding US patents
US
8,293,491 or US 8,658,385), PCT/AU2009/000869 (or in any one of the
corresponding
US patents US 8,597,643, US 9,328,155 or US 10,238,716), PCT/AU2010/001070 (or

in any one of the corresponding publications VVO/2011/020155, US 9,127,059, US

9,688,771, or US 10,053,508), and PCT/AU2010/001741 (or in any one of the
corresponding publications WO 2011/075789 or US 8,835,609) the entire contents
of
which are hereby incorporated by reference. Preferably the binding polypeptide

comprises the amino acid sequence of the CDRs of the VH and/or VL chain of
antibody
2-2-1 described in PCT/AU2010/001070 (or in any one of the corresponding US
patents
US 9,127,059, US 9,688,771, or US 10,053,508) or BPM09 described in
PCT/AU2007/001541 (or in corresponding US publication US 2010-0036101) and
produced by the hybridoma AB253 deposited with the European Collection of
Cultures
(ECACC) under Accession no. 06080101, W02013185010A1 or W02019056023.
Alternatively, the binding polypeptide of the CAR may comprise the amino acid
sequences of the CDRs of the antibody sdAbs 2-2-3, 2-472-2, or 2-2-12
described in
WO 2017/041143 (also published as US 2019/0365805), and WO 2019/222796
(corresponding to US application 17/057,060), incorporated herein by
reference.
[0184] The binding polypeptide of the CAR may comprise the amino acid sequence
of
the VH and/or VL chains of an antibody described in any one of:
PCT/AU2002/000061 or
PCT/AU2002/001204 (or in any one of the corresponding US patents US 7,326,415,
US
7,888,473, US 7,531,171, US 8,080,635, US 8,399,617, US 8,709,425, US
9,663,584,
or US 10,450,380), PCT/AU2007/001540 (or in corresponding US patent US
8,067,550), PCT/AU2007/001541 (or in corresponding US publication US 2010-
0036101), PCT/AU2008/001364 (or in any one of the corresponding US patents US
8,440,186, US 9,181,320, US 9,944,701 or US 10,597,451), PCT/AU2008/001365 (or
in
any one of the corresponding US patents US 8,293,491 or US 8,658,385),
PCT/AU2009/000869 (or in any one of the corresponding US patents US 8,597,643,
US
9,328,155 or US 10,238,716), PCT/AU2010/001070 (or in any one of the
corresponding
publications WO/2011/020155, US 9,127,059, US 9,688,771, or US 10,053,508),
and
PCT/AU2010/001741 (or in any one of the corresponding publications WO
2011/075789
or US 8,835,609) the entire contents of which are hereby incorporated by
reference.
Preferably the binding polypeptide comprises the amino acid sequence of the VH
and/or
VL chains of the antibody 2-2-1 described in PCT/AU2010/001070 (or in any one
of the
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corresponding US patents US 9,127,059, US 9,688,771, or US 10,053,508) or
BPM09
described in PCT/AU2007/001541 (or in corresponding US publication US 2010-
0036101) and produced by the hybridoma AB253 deposited with the European
Collection of Cultures (ECACC) under Accession no. 06080101, VV02013185010A1
or
W02019056023. Alternatively, the binding polypeptide of the CAR may comprise
the
amino acid sequences of the VH and/or VL chains of the antibody sdAbs 2-2-3, 2-
472-2,
or 2-2-12 described in WO 2017/041143 (also published as US 2019/0365805), and

WO 2019/222796 (corresponding to US application 17/057,060), incorporated
herein by
reference.
[0185] The binding polypeptide of the CAR may comprise the amino acid sequence
of
an antibody or fragment thereof described in any one of: PCT/AU2002/000061 or
PCT/AU2002/001204 (or in any one of the corresponding US patents US 7,326,415,
US
7,888,473, US 7,531,171, US 8,080,635, US 8,399,617, US 8,709,425, US
9,663,584,
or US 10,450,380), PCT/AU2007/001540 (or in corresponding US patent US
8,067,550), PCT/AU2007/001541 (or in corresponding US publication US 2010-
0036101), PCT/AU2008/001364 (or in any one of the corresponding US patents US
8,440,186, US 9,181,320, US 9,944,701 or US 10,597,451), PCT/AU2008/001365 (or
in
any one of the corresponding US patents US 8,293,491 or US 8,658,385),
PCT/AU2009/000869 (or in any one of the corresponding US patents US 8,597,643,
US
9,328,155 or US 10,238,716), PCT/AU2010/001070 (or in any one of the
corresponding
publications VVO/2011/020155, US 9,127,059, US 9,688,771, or US 10,053,508),
and
PCT/AU2010/001741 (or in any one of the corresponding publications WO
2011/075789
or US 8,835,609) the entire contents of which are hereby incorporated by
reference.
Preferably the binding polypeptide comprises the amino acid sequence of sdAb 2-
2-1
described in PCT/AU2010/001070 (or in any one of the corresponding US patents
US
9,127,059, US 9,688,771, or US 10,053,508) or antibody BPM09 described in
PCT/AU2007/001541 (or in corresponding US publication US 2010-0036101) and
produced by the hybridoma AB253 deposited with the European Collection of
Cultures
(ECACC) under Accession no. 06080101, W02013185010A1 or W02019056023.
Alternatively, the binding polypeptide may comprise the amino acid sequences
of sdAbs
2-2-3, 2-472-2, or 2-2-12 described in WO 2017/041143 (also published as US
2019/0365805), and WO 2019/222796 (corresponding to US application
17/057,060),
incorporated herein by reference.
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[0186] A "signal peptide" refers to a peptide sequence that directs the
transport and
localisation of the protein within a cell, e.g. to a certain cell organelle
(such as the
endoplasmic reticulum) and/or the cell surface.
[0187] Generally, an "antigen binding domain" refers to the region of the CAR
that
specifically binds to an antigen (and thereby is able to target a cell
containing the
antigen). The CARs of the invention may comprise one or more antigen binding
domains. Generally, the targeting regions on the CAR are extracellular. The
antigen
binding domain may comprise an antibody or an antibody binding fragment
thereof. The
antigen binding domain may comprise, for example, full length heavy chain, Fab

fragments, single chain Fv (scFv) fragments, divalent single chain antibodies
or
diabodies. Any molecule that binds specifically to a given antigen such as
affibodies or
ligand binding domains from naturally occurring receptors may be used as an
antigen
binding domain. Often the antigen binding domain is a scFv. Normally, in a
scFv the
variable regions of an immunoglobulin heavy chain and light chain are fused by
a
flexible linker to form a scFv. Such a linker may be for example the "(G,/S,),-
linker" and
variations thereof but the skilled person will appreciate that various linker
sequences
and formats may be used.
[0188] In some instances, it is beneficial for the antigen binding domain to
be derived
from the same species in which the CAR will be used in. For example, when it
is
planned to use it therapeutically in humans, it may be beneficial for the
antigen binding
domain of the CAR to comprise a human or humanised antibody or antigen binding

fragment thereof. Human or humanised antibodies or antigen binding fragments
thereof
can be made by a variety of methods well known in the art. The CAR as
disclosed
herein has an extracellular linker/label epitope binding domain as an antigen
binding
domain allowing it to bind indirectly via a target cell binding molecule as
disclosed
herein to an antigen expressed on a target cell.
[0189] "Spacer" or "hinge" as used herein refers to the hydrophilic region
that is
between the antigen binding domain and the transmembrane domain. The CARs of
the
invention may comprise an extracellular spacer domain but it is also possible
to leave
out such a spacer. The spacer may include e.g. Fc fragments of antibodies or
fragments
thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions
of
antibodies, accessory proteins, artificial spacer sequences or combinations
thereof. A
prominent example of a spacer is the CD8alpha hinge.
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[0190] The transmembrane domain of the CAR may be derived from any desired
natural or synthetic source for such a domain. When the source is natural, the
domain
may be derived from any membrane-bound or transmembrane protein. The
transmembrane domain may be derived for example from CD8alpha or CD28. When
the key signalling and antigen recognition modules (domains) are on two (or
even more)
polypeptides, then the CAR may have two (or more) transmembrane domains. The
splitting of key signalling and antigen recognition modules enables small
molecule-
dependent, titratable and reversible control over CAR cell expression (Wu et
al, 2015,
Science 350: 293-303) due to small molecule-dependent heterodimerising domains
in
each polypeptide of the CAR.
[0191] The cytoplasmic domain (or the intracellular signaling domain) of the
CAR is
responsible for activation of at least one of the normal effector functions of
the immune
cell in which the CAR is expressed. "Effector function" means a specialised
function of a
cell, e.g. in a T cell an effector function may be cytolytic activity or
helper cell activity
including the secretion of cytokines. The intracellular signalling domain
refers to the part
of a protein that transduces the effector function signal and directs the cell
expressing
the CAR to perform a specialised function. The intracellular signalling domain
may
include any complete, mutated or truncated part of the intracellular
signalling domain of
a given protein sufficient to transduce a signal that initiates or blocks
immune cell
effector functions.
[0192] The function of the intracellular domains may be pro- or anti-
inflammatory
and/or immunomodulatory, or a combination of such.
[0193] Prominent examples of intracellular signalling domains for use in the
CARs
include the cytoplasmic signaling sequences of the T cell receptor (TCR) and
co-
receptors that initiate signal transduction following antigen receptor
engagement.
[0194] Generally, T cell activation can be mediated by two distinct classes of

cytoplasmic signalling sequences, firstly those that initiate antigen-
dependent primary
activation through the TCR (primary cytoplasmic signalling sequences) and
secondly
those that act in an antigen-independent manner to provide a secondary or co-
stimulatory signal (secondary cytoplasmic signalling sequences, co-stimulatory

signalling domain). Therefore, an intracellular signalling domain of a CAR may
comprise
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one or more primary cytoplasmic signalling domains and/or one or more
secondary
cytoplasmic signalling domains.
[0195] Primary cytoplasmic signalling sequences that act in a stimulatory
manner
may contain ITAMs (immunoreceptor tyrosine-based activation motifs) signalling
motifs.
[0196] Examples of ITAM containing primary cytoplasmic signalling sequences
often
used in CARs are those derived from TCR zeta (003 zeta), FcR gamma, FcR beta,
CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d. Most
prominent is the sequence derived from CD3 zeta.
[0197] The cytoplasmic domain of the CAR may be designed to comprise the CD3-
zeta signaling domain by itself or combined with any other desired cytoplasmic

domain(s). The cytoplasmic domain of the CAR can comprise a CD3 zeta chain
portion
and a co-stimulatory signalling region. The co-stimulatory signalling region
refers to a
part of the CAR comprising the intracellular domain of a co-stimulatory
molecule. A co-
stimulatory molecule is a cell surface molecule other than an antigen receptor
or their
ligands that is required for an efficient response of lymphocytes to an
antigen. Examples
for a co-stimulatory molecule are 0D27, 0D28, 4-1BB (0D137), 0X40, CD30, CD40,

PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7,
LIGHT,
NKG2C and B7-H3.
[0198] The cytoplasmic signalling sequences within the cytoplasmic signalling
part of
the CAR may be linked to each other with or without a linker in a random or
specified
order. A short oligo-or polypeptide linker, which is preferably between 2 and
10 amino
acids in length, may form the linkage. A prominent linker is the glycine-
serine doublet.
[0199] As an example, the cytoplasmic domain may comprise the signalling
domain
of CD3-zeta and the signalling domain of CD28. In another example the
cytoplasmic
domain may comprise the signalling domain of CD3-zeta and the signalling
domain of
0D27. In a further example, the cytoplasmic domain may comprise the signalling

domain of CD3-zeta, the signalling domain of 0D28, and the signalling domain
of 0D27.
[0200] As aforementioned, either the extracellular part or the transmembrane
domain
or the cytoplasmic domain of a CAR may also comprise a heterodimerising domain
for
the aim of splitting key signalling and antigen recognition modules of the
CAR.
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[0201] Non-limiting examples of CARs that may be used in accordance with the
present invention are set forth in SEQ ID NOs: 165-167, 266 or 272. An example
of the
architecture of various CAR molecules is also provided herein in Figure 35.
Further
examples of CARs that may be used in accordance with the invention, are
provided in
WO 2017/041143 (also published as US 2019/0365805), and WO 2019/222796
(corresponding to US application 17/057,060), incorporated herein by
reference.
[0202] A CAR for use in accordance with the present invention, i.e. a CAR
comprising
an nfP2X7 E200 binding domain, may be designed to comprise any portion or part
of the
above-mentioned domains as described herein in any order and/or combination
resulting in a functional CAR.
[0203] The CARs as disclosed herein, or polypeptide(s) derived therefrom,
nucleic
acid molecule(s) or recombinant expression vectors cells encoding said CARs,
or
populations of cells expressing said CARs, may be isolated and/or purified.
The term
"isolated" means altered or removed from the natural state. For example, an
isolated
population of cells means an enrichment of such cells and separation from
other cells
that are normally associated in their naturally occurring state with said
isolated cells. An
isolated population of cells means a population of substantially purified
cells that are a
more homogenous population of cells than found in nature. Preferably, the
enriched cell
population comprises at least about 90% of the selected cell type. In
particular aspects,
the cell population comprises at least about 90%, 91%, 92%, 93%, 94%, 95%,
96%,
97%, 98%, 99%, or even 100% of the selected cell type.
[0204] The affinity at which the dysfunctional P2X7 receptor binding domain of
the
CAR binds to the nfP2X7 recognition site E200 of the bridging molecule can
vary, but
generally the binding affinity may be in the range of 100 pM, 1 nM, 10 nM, or
100 nM,
preferably at least about 1 pM or 10 pM, even more preferably at least about
100 pM.
[0205] CAR T cells targeted to EGFRvIll may be used to treat solid cancers.
EGFRvIll is a frequent splice variant of EGFR skipping exons 2-7. EGFRvIll is
tumour
specific and does not occur in healthy cells as EGFR is tightly regulated in
normal cells.
EGFRvIll is commonly expressed in glioblastoma but also in breast cancer and
head
and neck cancer. The EGFRvIl I-CAR T in this context may have the sequence
(SEQ ID
NO: 266) and is targeted to the epitope resulting out of the fusion of the
amino acid
sequence starting at position 25-29 LEEKK, followed by the insertion of G and
the
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subsequent amino acid sequence 298-304 NYVVTDH, the total epitope comprises or

consists of the sequence LEEKKGNYVVTDH (SEQ ID NO: 267). The complete EGFR
sequence is found at UniProtKB - P00533 (EGFR_HUMAN) and the complete protein
counts 1210 amino acids in isoform 1.
[0206] EGFRvIll targeted CAR T cells may be used to treat glioblastoma in a
conventional way to target EGFRvIll on cancer cells, but may also be
redirected to other
cancer-associated target antigens via the bridging molecules described herein
if the
EGFRvIll epitope moiety is integrated into the sequence of bridging molecules.
The
EGFRvIl I CAR T cells then can be used in the same manner as outlined for the
nfP2X7
CAR targeted approach described herein. The peptide tag may be the 13-mer
peptide
LEEKKGNYVVTDH or a shortened or extended natural or artificial variant
thereof, of
SEQ ID NO: 267.
[0207] The amino acid sequence of EGFRvIll CAR compatible bridging molecules
targeted to 0D33 and Her2 are described in Table 1 as SEQ ID NO: 268 and 269,
and
SEQ ID NO: 270 and 271, respectively.
[0208] CLDN6 targeted CAR T cells may be used to treat solid cancers e.g.
ovarian
cancer. The CLDN6-CAR T in this context may have the sequence (SEQ ID NO: 272)

and is targeted to the second extracellular domain of CLDN6 [UniProtKB -
P56747
(CLD6_HUMAN] cells directly via the amino acid sequence [ECD2, >spIP567471138-
160 VVTAHAIIRDFYNPLVAEAQKREL (SEQ ID NO: 273)] but may also be redirected to
other cancer-associated target antigens, e.g. CD33 or Her2 via the bridging
molecules
described herein, if the CLDN6 epitope moiety is integrated into the sequence
of
bridging molecules. The CLDN6 CAR T cells then can be used in the same manner
as
outlined for the nfP2X7 CAR targeted approach described herein. The peptide
tag may
be the 23-mer peptide VVTAHAIIRDFYNPLVAEAQKREL or a shortened or extended
natural or artificial variant thereof, such as SEQ ID NO: 274 or 275.
Bridging molecule
[0209] It will be appreciated that the bridging molecule may be in any form,
provided
that it comprises a) a targeting moiety for binding a target cell and b) a
tumour-specific
antigen epitope moiety. Preferably, the tumour-specific antigen epitope moiety
is, or
comprises, a dysfunctional P2X7 receptor epitope moiety, a EGFRvIll epitope
moiety or
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a CLDN6 epitope moiety, preferably such that the epitope moiety is recognised
by the
tumour-specific CAR which is being redirected, in accordance with the methods
of the
invention.
[0210] Typically, the targeting moiety is in the form of a fusion protein in
which the
targeting moiety is linked to the tumour-specific antigen epitope moiety,
preferably
dysfunctional P2X7 receptor epitope moiety, directly or via a linker.
[0211] It will be appreciated that the bridging molecule may have any of a
different
number of architectures. For example, and as described further herein, the
targeting
moiety for binding a target cell may be in the form of any suitable binding
domain, such
as derived from an antibody, or fragments thereof. The targeting moiety may be
linked
to the tumour-specific antigen epitope moiety in any configuration. For
example, the
tumour-specific antigen epitope moiety may be linked to the N- or C-terminus
of the
targeting moiety. Examples of suitable architectures are provided in the
Figures herein.
In particularly preferred embodiments, the tumour-specific antigen epitope
moiety is
linked to the targeting moiety via its C terminal region, such that the N-
terminal region of
tumour-specific antigen epitope moiety is freely accessible for binding by the
tumour-
specific CAR.
[0212] Any suitable linker may be used for linking targeting moiety to the
tumour-
specific antigen epitope moiety. The linker may comprise a polypeptide, a
peptide or a
chemical group.
[0213] A linker may be a peptide having a length of up to 20 amino acids. The
term
"linked to" or "fused to" refers to a covalent bond, e.g., a peptide bond,
formed between
two moieties. Accordingly, in the context of the present invention the linker
may have a
length of 1,2, 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21 0r22
amino acids. For example, the herein provided bridging molecule may comprise a
linker
between the targeting moiety and tumour-specific antigen epitope moiety,
preferably the
dysfunctional P2X7 receptor epitope moiety. Such linkers have the advantage
that they
can make it more likely that the different polypeptides of the fusion protein
fold
independently and behave as expected.
[0214] The skilled person will be familiar with the design and use of various
peptide
linkers comprised of various amino acids, and of various lengths, which would
be
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suitable for use as linkers in accordance with the present invention. The
linker may
comprise various combinations of repeated amino acid sequences. The linker may
be a
flexible linker (such as those comprising repeats of glycine and serine
residues), a rigid
linker (such as those comprising glutamic acid and lysine residues, flanking
alanine
repeats) and/or a cleavable linker (such as sequences that are susceptible by
protease
cleavage).
[0215] The peptide linker may be any one or more repeats of Gly-Ser (GS), Gly-
Gly-
Ser (GGS), Gly-Gly-Gly-Ser (GGGS) or Gly-Gly-Gly-Gly-Ser (GGGGS) or variations

thereof. In any embodiment, the linker may comprise or consist of the sequence

GGGGSGGGGSGGGGS, i.e. (G4S)3.
[0216] In any embodiment, the peptide linker can include the amino acid
sequence
GGGGGS (a linker of 6 amino acids in length) or even longer. The linker may be
a
series of repeating glycine and serine residues (GS) of different lengths,
i.e., (GS)n
where n is any number from 1 to 15 or more. For example, the linker may be
(GS)3 (i.e.,
GSGSGS) or longer (CS)11 or longer. It will be appreciated that n can be any
number
including 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11 or more.
[0217] In further embodiments, the linker may comprise inclusion of an amino
acid
that provides rigidity, such as lysine. For example, in certain embodiments,
the linker
region may also comprise the sequence GSGK.
[0218] The peptide linker may consist of a series of repeats of Thr-Pro (TP)
comprising one or more additional amino acids N and C terminal to the repeat
sequence. For example, the linker may comprise or consist of the sequence
GTPTPTPTPTGEF (also known as the TP5 linker). In further aspects, the linker
may be
a short and/or alpha-helical rigid linker (e.g. A(EAAAK)3A, PAPAP or a
dipeptide such
as LE or CC).
[0219] In further embodiments, as an alternative or in addition to a glycine-
serine-
based linker region as described above, the targeting moiety may be in the
form of a
fusion protein in which the targeting moiety is linked to the tumour-specific
antigen
epitope moiety, preferably dysfunctional P2X7 receptor epitope moiety, via a
hinge
region. The linking between the targeting moiety and tumour-specific antigen
epitope
moiety may comprise a combination of hinge region and linker regions.
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[0220] Examples of suitable hinge regions include hinge regions derived from
immunoglobulins. The hinge region may be derived from an IgG1, IgG2, IgG3 or
IgG4,
and may comprise one or more amino acid substitutions, (for example to prevent
or
reduce the likelihood of disulphide bridge formation). Alternative hinge
sequences may
be derived from alternative immunoglobulin domains, CD8A, CD8B, CD4 or CD28,
TRAC, TRBC, TRGC, TRDC.
[0221] Table 2 below provides non-limiting examples of suitable hinge regions
for use
in joining the targeting moiety and tumour-specific antigen epitope moiety, in
the
bridging molecules of the invention.
[0222] It will be appreciated that the targeting moiety may be joined to the
tumour-
specific antigen epitope moiety by more than one linker and/or more than one
hinge
region. For example, the fusion protein may comprise (N to C terminus), the
tumour-
specific antigen epitope moiety, conjugated directly to the targeting moiety.
Alternatively, the fusion protein may comprise the tumour-specific antigen
epitope
moiety, followed by a linker region, then the targeting moiety. Further still,
the fusion
protein may comprise the tumour-specific antigen epitope moiety, followed by a
linker
region, then a hinge region, and then the targeting moiety. In a further
embodiment still,
the fusion protein may comprise the tumour-specific antigen epitope moiety,
followed by
a linker region, then a hinge region, a further linker region, then the
targeting moiety. Of
course, the skilled person will appreciate that the alternative configuration
is possible (ie
wherein the tumour-specific antigen epitope moiety is joined to the C terminus
of the
targeting moiety, via one or more linker and/or hinge regions.
[0223] Table 2: exemplary hinge regions
Amino acid sequence SEQ ID NO: Desc ription
UMSSIAtinSPRS.RMINVISENCOnigiiiiintlettlarg$:,..MMai5iii.T;iqi5FOr.0519,91i"
EPKSSDKTHTSPP 311
'EPKSSDKTHT 313
D KTH TS PP 315
EPKSDKTHTSPPSP 3177
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EP KS G D KTH TG PP 319
EPk.8.0',:-(THrTt!....Y.PPP:::::::.F.T.:::::V....1.:::.::::::.::
:.i!:::.:::.::!:::.::::.!.:1;261..i!::..5.!M::1!:::.:::::.::5:::.:::::!::::.!::
::::i!::::1!:::.:::.::!:::.:::.::::::::.::!:::.1:"
!:::::::::::::::=:::::=::::::::::::::::::::::=::=::::::::::::::::::::=::::::::=
::=:::::::::::::: :=::=::=::=::=::=::=:::=::::::::::::::: :=::=::=:::=:=::=:::
[-:-.PKScit)ic. T i -1-1 321

i:=::=:=:=::::=:=:==:::=:=::::=:=::::=:=:==:=:=:=::=:=:=::::=:=:==:=:=::::=:=:=
::=:=:=:=::=:=::::=:=:=::=:=:=::::=:=:=::=:=:=::=:=:=:=:=:=:=:.
=:.::.:.:.?.?.?.?.?...?..:...::.:.::?.?.?.u....u..:-..:...f.?.???????.:????-
???????.:;.???????-???.:??????.:i.
=:::.:.::i:.::::=:=:=:==:=:=:=::=:=:,:=:=:==:=:=:=::=:=:=::=:=:=:::::::::::::::
:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::.:
_ ..................... = ========.= ====== ===== ===========
..................... ===== =======_===,.....= ============== =============
...= ============= ============= ============== ============= ===
============= ============ .============ =========
0 K TH .1-G P1-'(.;:v
= = "
= : =i==i=ii=i=i=ii=i-i=i-i=i=i=i=ii=ii=i=i=i-i-i=i-
i=:=:=::=:=:=:=:=:=i=i-i-
i=i=ii=:=:=:,=:=::=:==i=i=i=i=i::*::i..:...:.::...:.:::::::i::...:.::.:.:::::..
.:.::...:.uj::::...:.::...:.::...:.::...:.in::.::::::::::::::.
.............. . .
323 '...
====:::::==== ::::::::: :==
::.=:.=:.==:õ:õ:õ:õi=:õ:õ:.:.=:.=:.=:õ.:.=:.=:.=:õ:õ:õ:õ:.i.:õ:õ:õ...:=:=:=.:=:
=:=::=:=:=:=:=:=:=:.
EPKSDKT-i TP.Pi-
7.i..i.:.:.,:.:..::.:.:.:..:.:::.:.:.:.::.:..:,.:.:.:..:.:::.:**`,344;=.:i:::::
:izi:....m::::i:m:::m:i:i:m::...:.:::.:...:.:.:i::...:.::!::...:.::...:.::...:.
..:.:.::.::...:.:::...:...:.::!::...:...:.:...:.::...:...:.::...:...:.:::...:..
.:...:.!::...:.::...:.::...:...:.:::...:...:.::...:.::...:...:.!::...:...:.:::.
..:...:.:::.:.::...:...:.
= . =
::. :
3:::;6:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
::::::::::::::::::::=:ggMEMn:Miffg%.*=.g
E-: P iSS(.31)K-1-:--1 T Z-3 I-) P S P

.........,...................,.........,.......................................
....,..........................................,.........,..........,..........
..........,................................
. ............ ............ ... ............
.....................................
=:===:=====::=:::=:===:===:=:==i===:===:====i===:==ff==:===:===::===:=====:===:
===:====i===:===:===:=:===:==H===:===:=====:===:===:===:i===:===:===:===:===:==
=:===:=====:===:=====:===:===:i===:===:===:=====:===:==:==::=:==:==:=*:
EPKSGE1k-(T-ITG PP SP " = 326.i-
..;::=.;.::..;...=.....................i.............:-
..........................................i............:-
........................................=....i..........; i-
i...;..:.:..:..:.:..i.:..:...i.:..:..:..::::..:.:::..:..:...:..:..:...i.:..:..:
.::::..:....:..:..:..:.i.:..:..:...:..:..:..:::...:..:..:...:...::.:::::
i :
.i!i.ii.ei.ii.iiSii.iMiii.ii.ffiii.iNii.ffiii.ai!iiii
.Eiiiiiii.Hi=MiaiiiiiMaiai.RMiNii.ffii ai .i.i.:.
=
C-:-.PKSSE)K-I-17::::=Pc? ,P 327

======::=:=:=::::=:=:==:::=:=::::=:=::::=:=:==:=:=:=::=:=:=::::=:=:==:=:=::::=:
=:=::=:=:=:=::=:=::::=:=:=::=:=:=::::=:=:=::=:=:=::=:=:=:=:=:=:=:.
_.=
........... ............
...............................................................................
...............
...............................................................................
... ',..?====:..n*:-.*::?.*:=::i:?.*:::;:u..*::?.*:=:-
.*::?.*:::?.?.*:::;:=:::::::::::::::::::::::::::::::::::::::::::::::::::::,.:::
::::::::::::::::::::::::::::
EpKssDKTHTGppSp:0::=i=ig:i:=i:T.:*i::=i:=i:=i::=i:=ifi:=i=i42:g::=i:=i=i:=i:=i:
=i=i:=ifi:=i::=i::=i:=i::=i:=i=i:=i:=i:=i::=ifi:=i::=i::=i:=i=i::=i:=ifi:=i:=i:
=i::=ifi:=i:=i=i::=i:=ifi::i.=i....i..:.:.=::1:::i::g
=:=:=:=::=:=:=::i:=:=:==:=:=;=:=:=::=:=:=:=i:=:=:=:=:=::=:=:=:gi:=:=:=::=:=g
=:*
.... ............ ............ ............ ................ ............
: .

=:i*:.:.:*:g.:*:g*:n.:*:.:*:*.g*:.:*:g:=:.:.:*:.:*:i*:=:.:*:Mi.:*:.:M=:.:*:i*:=
:.:*:i*:.:*:.:.:*:.:*:.:*:i*:.:*:.:.:*:i*:.:*:.:M=:Mi*:.:.:*:i*:.:*:P.:.:*:i*:.
:*:.:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::s:::::::
::::::::::::::::s:::::::::::::::::::::::::::::::::::::::::::::::.:1
EPi<SSDK-11-11-GPP 329
::=:.:*:?:i.ai...:**M.:*:.:.:i..:**:.:**ffl.Mi..:*:.:*:ni*:.:*:.:*:.:?:=:i..:*:
.:*:M.:i*:.:*:.:*:=:.:*:.:*:.:*:::=:.:*:.:*:.:
EPKBaD KT:-ITaP: P
.&:::::::::::A::::::::::::::.:..:.:3.,1Ci.::.:.:.:.::.:.:i::::.:.:::.:.::.:.:i:
:=:.:.:::.:.:N::i:.:.::::.:.:i:.=::.:.:.:.:.:..:.:.:.:.:.:.:.::.:.:.:.:.:.:::.:
.:.:.:.:..:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.::.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.
:.:.:.::.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.::.:.:.:.:.:.:.:.:.:.:.:.:.:
.:.:.
F:-.1:-) K .-31.1.) K. I- hi -1.I.PP. -IP 331
....:: :.:.:..:.:.:,:.:.:.::.:.:.:..:.:.:;.:.:.:.::.:.:.:.:.:.:.:..:.:.:
::.:.= = = = -
=.:.:.::.:.:.:::::::::::::::::::::::::::i:i:iõi:i**::::::::::::::::::::::::::::
::::::::::::
==::=:.:;.:.:=::=::.:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
::::::::::: ::::::::::::::::::::::::::::::: :::::::::::::::::::::::
..EF3.K8170:KTH T.T.PPi:i::. :::::::::i::.n::Z:::. ::i::::::.:41..3'2:i:
..........
::.:.==:.==:..i.:.=:.=:..i.:.=:.:.=:::=:==:::=:==:=i=:.=:.:..i.:õ:õ...:.===:.=:
.:.=i=:.=:õ i i.:.===:.=:.=:.=:.:..i.:.=:...i.:.=:.=:.=:.=:.=:.=:.
=:.=:.==:.=:.=:.== =:õ:õ:.:.=:::::::::::::.==:õ:õ:õ:.
=:õ:õ:õ...::::::::õ:õ:õ:õ:õ:õ:õ:. =:õ:õ:õ=:õ:õ:õ:,:õ:õ:õ:.
??i.:.???.???.???::.:..:.i.????.???.::.????:.:.:..::.??.???.???????.::.???.??.:
i.???.::.???????.???.::.???????.???.::.??.::.???.?i.?????.::.???.???.?i.?.::.??
?????.???.::.i.???.......:::::::................:...........:..................
.............-........................................................-
.................................??????-???????.: .???.?:?.:?..i
EPKSTDK-1H-1-1-PPP 333

:,.:::::::========,.:,.:,.,.:i:,.:,.:*,.:,.:,.:,.:,.:,.:,.,.:,.:,.:,.::,.:,.:,.
:::,.:,.:,.,.:,.:,.:,..:,.:,.:,.::,.:,.:,.:,.
:,.::.::.s.::.::.::.:::.::.::.::.::.::.::.::. ::.::.::.:::.::::::::::=======
=:.=i=======!=============================!=========================!==========
=== ==============!============= =============!=======
EP KS'i'D Ki H 'Ã' ,),-i
:
,, = .=:-
:=:==:::=:==:=:=:=::=:=:=:s::::::::::::::::::::::::::::::::::::::::::::::::::::
:::::::::::::: i:i i:i::::::: ::::::::: i:i:i::i:i:i::i:i:ii:::::::
:::::::::::::::::::::::.:. :j:j:j :j:.:.::.::.::.::.:
= k.i=ii=i
.i*.:.:**:=:.:**:***:1*.i=fi==i=i i*i :=:**:=::=:**:=i=i=i ff i=i=i*i
:=:**:=::=:=::=i=i=i=i===i***: f::: : :: : : : i i fi : : : ::: : : : : :
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EPKSXDTF-)PPXPR 358 Mutated IgG3
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KYGPPCPXCP 364 Mutated IgG4
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Targeting moiety of the bridging molecule
[0224] The targeting moiety of the bridging molecule may bind to a cell
surface
molecule on a target cell. The cell surface molecule may comprise an antigen.
The cell
surface molecule may be selected from a protein, a lipid moiety, a
glycoprotein, a
glycolipid, a carbohydrate, a polysaccharide, a nucleic acid, an MHC-bound
peptide, or
a combination thereof. The cell surface molecule may comprise parts (e.g.,
coats,
capsules, cell walls, flagella, fimbriae, and toxins) of bacteria, viruses,
and other
microorganisms. The cell surface molecule may be expressed by the target cell.
The
cell surface molecule may not be expressed by the target cell. By way of non-
limiting
example, the cell surface molecule may be a ligand expressed by a cell that is
not the
target cell and that is bound to the target cell or a cell surface molecule of
the target cell.
Also, by non-limiting example, the cell surface molecule may be a toxin,
exogenous
molecule or viral protein that is bound to a cell surface or cell surface
receptor of the
target cell.
[0225] The bridging molecules may interact with a plurality of target cells.
The target
cell may be an infected cell. The target cell may be a pathogenically infected
cell. The
target cell may be a diseased cell. The target cell may be a genetically
modified cell.
The target cell may not be a host cell. The target cell may come from an
invading
organism (e.g. yeast, worm, bacteria, fungus). Further disclosed herein are
bridging
molecules that interact with a molecule on a non-cell target. The non-cell
target may be
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a virus or a portion thereof. The non-cell target may be a fragment of a cell.
The non-cell
target may be an extracellular matrix component or protein.
[0226] The target cell may be derived from a tissue. The tissue may be
selected from
brain, oesophagus, breast, gut, intestine, colon, lung, glia, ovary, uterus,
testes,
prostate, gastrointestinal tract, bladder, liver, spleen, thymus, bone, fat
and skin. The
target cell may be derived from one or more endocrine glands. Alternatively,
or
additionally, the target cell may be derived from one or more endocrine
glands. The
endocrine gland may be a lymph gland, pituitary gland, thyroid gland,
parathyroid gland,
pancreas, gonad or pineal gland.
[0227] The target cell may be selected from a stem cell, a pluripotent cell, a

hematopoietic stem cell or a progenitor cell. The target cell may be a
circulating cell.
The target cell may be an immune cell.
[0228]
The target cell may be a cancer stem cell. The target cell may be a cancer
cell. The cancer cell may be derived from a tissue. The tissue may be selected
from, by
way of non-limiting example, a brain, an oesophagus, a breast, a colon, a
lung, a glia,
an ovary, a uterus, a testicle, a prostate, a gastrointestinal tract, a
bladder, a liver, a
thyroid and skin. The cancer cell may be derived from bone. The cancer cell
may be
derived from blood. The cancer cell may be derived from a B cell, a T cell, a
monocyte,
a thrombocyte, a leukocyte, a neutrophil, an eosinophil, a basophil, a
lymphocyte, a
hematopoietic stem cell or an endothelial cell progenitor. The cancer cell may
be
derived from a CD19-positive B lymphocyte. The cancer cell may be derived from
a
stem cell. The cancer cell may be derived from a pluripotent cell. The cancer
cell may
be derived from one or more endocrine glands. The endocrine gland may be a
lymph
gland, pituitary gland, thyroid gland, parathyroid gland, pancreas, gonad or
pineal gland.
[0229] The cell surface molecule of the target cell may be a receptor_ The
receptor
may be an extracellular receptor. The receptor may be a cell surface receptor.
By way
of non-limiting example, the receptor may bind a hormone, a neurotransmitter,
a
cytokine, a growth factor or a cell recognition molecule. The receptor may be
a
transmembrane receptor. The receptor may be an enzyme-linked receptor. The
receptor may be a G-protein couple receptor (GPCR). The receptor may be a
growth
factor receptor. By way of non-limiting example, the growth factor receptor
may be
selected from an epidermal growth factor receptor, a fibroblast growth factor
receptor, a
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platelet derived growth factor receptor, a nerve growth factor receptor, a
transforming
growth factor receptor, a bone morphogenic protein growth factor receptor, a
hepatocyte growth factor receptor, a vascular endothelial growth factor
receptor, a stem
cell factor receptor, an insulin growth factor receptor, a somatomedin
receptor, an
erythropoietin receptor and homologs and fragments thereof. The receptor may
be a
hormone receptor. The receptor may be an insulin receptor. By way of non-
limiting
example, the receptor may be selected from an eicosanoid receptor, a
prostaglandin
receptor, an oestrogen receptor, a follicle stimulating hormone receptor, a
progesterone
receptor, a growth hormone receptor, a gonadotropin-releasing hormone
receptor,
homologs thereof and fragments thereof. The receptor may be an adrenergic
receptor.
The receptor may be an integrin. The receptor may be an Eph receptor. The
receptor
may be a luteinising hormone receptor. The cell surface molecule may be at
least about
50% homologous to a luteinising hormone receptor. The receptor may be an
immune
receptor. By way of non-limiting example, the immune receptor may be selected
from a
pattern recognition receptor, a toll-like receptor, a NOD-like receptor, a
killer-activated
receptor, a killer inhibitor receptor, an Fc receptor, a B cell receptor, a
complement
receptor, a chemokine receptor and a cytokine receptor. By way of non-limiting

example, the cytokine receptor may be selected from an interleukin receptor,
an
interferon receptor, a transforming growth factor receptor, a tumour necrosis
factor
receptor, a colony stimulating factor receptor, homologs thereof and fragments
thereof.
The receptor may be a receptor kinase. The receptor kinase may be a tyrosine
kinase
receptor. The receptor kinase may be a serine kinase receptor. The receptor
kinase
may be a threonine kinase receptor. By way of non-limiting example, the
receptor
kinase may activate a signalling protein selected from a Ras, a Raf, a PI3K, a
protein
kinase A, a protein kinase B, a protein kinase C, an AKT, an AMPK, a
phospholipase,
homo logs thereof and fragments thereof. The receptor kinase may activate a
MAPK/ERK signalling pathway. The receptor kinase may activate Jak, Stat or
Smad.
[0230] The cell surface molecule may be a non-receptor cell surface protein.
The cell
surface molecule may be a cluster of differentiation proteins. By way of non-
limiting
example, the cell surface molecule may be selected from CD3, CD4, CD8, CD11a,
CD11b, CD13, CD14, CD15, CD16, CD22, CD24, CD25, C030, CD31, CD33, CD34,
0D38, 0D45, C056, CD61, CD91, CD114, CD117, CD182, CD200, fragments thereof,
and homologs thereof.
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[0231] The cell surface molecule of the target cell may be a
molecule that does not
comprise a peptide. The cell surface molecule may comprise a lipid. The cell
surface
molecule may comprise a lipid moiety or a lipid group. The lipid moiety may
comprise a
sterol. The lipid moiety may comprise a fatty acid. The antigen may comprise a

glycolipid. The cell surface molecule may comprise a carbohydrate.
[0232] The cell surface molecule of the target cell may be an antigen. The
antigen
may be at least a portion of a surface antigen or a cell surface marker on a
cell. The
antigen may be a receptor or a co-receptor on a cell. The antigen may refer to
a
molecule or molecular fragment that may be bound by a major histocompatibility

complex (MHC) and presented to a T-cell receptor. The term "antigen" may also
refer to
an immunogen. The immunogen may provoke an adaptive immune response if
injected
on its own into a subject. The immunogen may induce an immune response by
itself.
The antigen may be a superantigen, T-dependent antigen or a T-independent
antigen.
The antigen may be an exogenous antigen. Exogenous antigens are typically
antigens
that have entered the body from the outside, for example by inhalation,
ingestion, or
injection. Some antigens may start out as exogenous antigens, and later become

endogenous (for example, intracellular viruses). The antigen may be an
endogenous
antigen. The endogenous antigen may be an antigen that has been generated
within
cells as a result of normal cell metabolism, or because of pathogenic
infections (e.g.,
viral, bacterial, fungal, parasitic). The antigen may be an autoantigen. The
autoantigen
may be a normal protein or complex of proteins (and sometimes DNA or RNA) that
is
recognised by the immune system of patients suffering from a specific
autoimmune
disease. These antigens should, under normal conditions, not be the target of
the
immune system, but, due to genetic and/or environmental factors, the normal
immunological tolerance for such an antigen is not present in these patients.
The
antigen may be present or over-expressed due to a condition or disease. The
condition
or disease may be a cancer or a leukaemia. The condition may be an
inflammatory
disease or condition. The condition or disease may be a metabolic disease. The

condition may be a genetic disorder.
[0233] The present invention also may find application for the
treatment of specific B-
or T-lineage associated autoimmune diseases, for example using anti-idiotypic
antibodies or fragments thereof or ligands thereof for targeting the B cell
receptor and/or
the T cell receptor. Such diseases include myasthenia gravis, systemic lupus
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erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), solid
organ
transplant hyperacute, acute, chronic or mix-type rejection, bone marrow or
stem cell
transplant rejection, and graft versus host disease.
[0234] The cell surface molecule of the target cell may be an antigen that has
been
designated as a tumour antigen. Tumour antigens or neo-antigens may be
antigens that
are presented by MHC I or MHC II molecules on the surface of tumour cells.
These
antigens may sometimes be presented by tumour cells and never by the normal
ones.
In this case, they are called tumour-specific antigens (TSAs) and, in general,
result from
a tumour-specific mutation. More common are antigens that are presented by
tumour
cells and normal cells, and they are called tumour-associated antigens (TAAs).
A TAA
associated antigen is not unique to a tumour cell and instead is also
expressed on a
normal cell under conditions that fail to induce a state of immunologic
tolerance to the
antigen. The expression of the antigen on the tumour may occur under
conditions that
enable the immune system to respond to the antigen. TAAs may be antigens that
are
expressed on normal cells during foetal development when the immune system is
immature and unable to respond or they may be antigens that are normally
present at
extremely low levels on normal cells but which are expressed at much higher
levels on
tumour cells. Cytotoxic T lymphocytes that recognise these antigens may be
able to
destroy the tumour cells before they proliferate or metastasise. Tumour
antigens may
also be on the surface of the tumour in the form of, for example, a mutated
receptor, in
which case they may be recognised by B cells.
[0235] Non-limiting examples of TSA or TAA antigens include the following:
Differentiation antigens such as MART-1/MelanA (MART-I), gp 100 (Pmel 17),
tyrosinase, TRP-1, TRP-2 and tumour-specific multilineage antigens such as
MAGE-1,
MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as
CEA; overexpressed oncogenes and mutated tumour-suppressor genes such as p53,
Ras, HER-2/neu; unique tumour antigens resulting from chromosomal
translocations
such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such
as
the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV)
antigens E6
and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5,
MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA
19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16,
43-
9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HOG, BCA225, BTAA, CA 125, CA 15-
3\CA
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27.29\BCAA, CA 195, CA 242, CA-50, CAM43, 20 CD68\PI, CO-029, FGF-5, G250,
Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCASI,
SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6,

TAG72, TLP, and TPS.
[0236] The cell surface molecule of the target cell may be an antigen selected
from
the group consisting of any surface expressed antigens. Exemplary target
antigens may
comprise but are not limited to: CD33 (Siglec-3), CD123 (IL3RA), CD135 (FLT-
3), CD44
(HCAM), CD44V6, CD47, CD184 (CXCR4), CLEC12A (CLL1), LeY, FR, MICA/B,
CD305 (LAIR-1), CD366 (TIM-3), CD96 (TACTILE), CD133, CD56, CD29 (ITGB1),
CD44 (HCAM), CD47 (IAP), CD66 (CEA), CD112 (Nectin2), CD117 (c-Kit), CD133,
CD146 (MCAM), CD155 (PVR), CD171 (L1CAM), CD200 (OX-2), CD221 (IGF1),
CD227 (MUC1), CD243 (MRD1), CD246 (ALK), CD271 (LNGFR), CD19, CD20, GD2,
and EGFR. Other target antigens include TCR, sugars, lipids, carbohydrates or
any
other molecule expressed on the surface of the target cell. The antigen may be
any
antigen referred to in Table 1 in the context of a bridging molecule or
binding construct.
[0237] In preferred embodiments, the target cell is a cancer cell and the cell
surface
molecule of the cancer cell is an antigen that is associated with the cancer.
The antigen
may be a tumour-specific antigen or a tumour-associated antigen. The antigen
may be
one which is associated with a particular type of cancer. For example,
overexpression of
the antigen may be associated with a specific cancer or specific class of
cancer. For
example, where the cancer is breast cancer, the antigen may be associated with
breast
cancer but not with another form of cancer. Alternatively, the antigen may be
associated
with a class of cancers such as solid tumours, but not associated with
haematological
(ie "liquid") tumours, or vice versa. The antigen may be associated with
cancers of a
particular lineage but not with others. For example the antigen may be
associated with
sarcomas but not lymphomas or carcinoma. As used herein the term "associated
with"
in relation to cancer, will be understood to mean that the antigen's
expression (whether
increased or decreased) is considered a marker of the cancer. It will be
appreciated that
there may be low levels of expression of an antigen but this does not equate
to the
antigen being "associated" with a given cancer.
[0238] Suitable cancer antigens which may be bound by the targeting moiety of
the
bridging molecule include, but are not limited to, mesothelin (MSLN), prostate
specific
membrane antigen (PSMA), prostate stem cell antigen (PCSA), carbonic anhydrase
IX
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(CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22,
CD30,
CD33, CD34, CD38, CD41, CD44, CD49f, 0D56, CD74, 0D123, CD133, CD138,
epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40),
epithelial cell
adhesion molecule (EpCAM), folate-binding protein (FBP), foetal acetylcholine
receptor
(AChR), folate receptor-a and 13 (FRa and 8), Ganglioside G2 (GD2),
Ganglioside G3
(GD3), human Epidermal Growth Factor Receptor 2 (HER-2/ERB2), HER3, Epidermal
Growth Factor Receptor vlIl (EGFRvIII), ERB3, ERB4, human telomerase reverse
transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (1L-13Ra2), k-
light chain,
kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell
adhesion molecule (L1CAM), melanoma-associated antigen 1 (melanoma antigen
family Al, MAGE-A1), Mucin 16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands, cancer-

testis antigen NY-ESO-1, oncofoetal antigen (h5T4), tumour-associated
glycoprotein 72
(TAG-72), vascular endothelial growth factor R2 (VEGF- R2), Wilms' tumour
protein
(WT-1), type 1 tyrosine-protein kinase transmembrane receptor (ROR1), B7-H3
(CD276), B7-H6 (Nkp30), Chondroitin sulfate proteoglycan-4 (CSPG4), DNAX
Accessory Molecule (DNAM-1), Ephrin type A Receptor 2 (EpHA2), Fibroblast
Associated Protein (FAP), Gp100/HLA-A2, Glypican 3 (GPC3), HA-1H, HERK-V, IL-1

1Ra, Latent Membrane Protein 1 (LMP1), Neural cell-adhesion molecule (N-
CAM/C056), and Trail Receptor (TRAIL R). It is understood that these or other
cancer
antigens can be utilised for targeting by a bridging molecule in the present
invention.
[0239] The targeting moiety of the bridging molecule may be any binding
molecule,
for example, a full-size antibody, or fragment thereof, or any antibody or
fragment
thereof described herein, an immunocytokine (antibody linked to a cytokine, or

fragments thereof), a ligand (protein related, peptides, sugar molecules,
processed
molecules, lipids, cytokines, hormones), a soluble T cell receptor (TcR), a
single chain
(Sc) TcR, single chain T cell receptor binding motifs and a T cell receptor
like mAb, an
aptamer (such as DNA or RNA), a peptide (e.g. aptamers or bicyclic peptides),
a toxin,
a lipid or a carbohydrate.
[0240]
The targeting moiety of the bridging molecule may be a polypeptide and may
be a targeting antibody or antibody fragment. The targeting antibody or
antibody
fragment may be an immunoglobulin (Ig). The immunoglobulin may be selected
from an
IgG, an IgA, an IgD, an IgE, an IgM, a fragment thereof or a modification
thereof. The
immunoglobulin may be IgG. The IgG may be IgG1. The IgG may be IgG2. The IgG
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may be IgG3. The IgG may be IgG4. The IgG may have one or more Fc mutations
for
modulating endogenous T cell FcR binding to the bridging molecule. The IgG may
have
one or more Fc mutations for removing the Fc binding capacity to the FcR of
FcR-
positive cells. The one or more Fc mutations may remove a glycosylation site.
The one
or more Fc mutations may be selected from E233P, L234V, L235A, delG236, A327G,

A330S, P331S, N2970 and any combination thereof. The one or more Fc mutations
may be in IgG1. The one or more Fc mutations in the IgG1 may be L234A, L235A,
or
both. Alternatively, or additionally, the one or more Fc mutations in the IgG1
may be
L234A, L235E, or both. Alternatively, or additionally, the one or more Fc
mutations in
the IgG1 may be N297A. Alternatively, or additionally, the one or more
mutations may
be in IgG2. The one or more Fc mutations in the IgG2 may be V234A, V237A, or
both.
[0241] The targeting antibody or antibody fragment may be an Fc null
immunoglobulin or a fragment thereof.
[0242] As used herein, the term "antibody fragment" refers to any form of an
antibody
other than the full-length form. Antibody fragments herein include antibodies
that are
smaller components that exist within full-length antibodies, and antibodies
that have
been engineered. Antibody fragments include, but are not limited to, Fv, Fc,
Fab, and
(Fab')2, single chain Fv (scFv), diabodies, triabodies, tetrabodies,
bifunctional hybrid
antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions,
framework
regions, constant regions, heavy chains, light chains, alternative scaffold
non-antibody
molecules, and bispecific antibodies. Unless specifically noted otherwise,
statements
and claims that use the term "antibody" or "antibodies" may specifically
include
"antibody fragment" and "antibody fragments."
[0243] The targeting antibody fragment may be human, fully human, humanised,
human engineered, non-human, and/or chimeric antibody. The non-human antibody
may be humanised to reduce immunogenicity to humans, while retaining the
specificity
and affinity of the parental non-human antibody. Chimeric antibodies may refer
to
antibodies created through the joining of two or more antibody genes that
originally
encoded for separate antibodies. A chimeric antibody may comprise at least one
amino
acid from a first antibody and at least one amino acid from a second antibody,
wherein
the first and second antibodies are different. At least a portion of the
antibody or
antibody fragment may be from a bovine species, a human species, or a murine
species. At least a portion of the antibody or antibody fragment may be from a
rat, a
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goat, a guinea pig or a rabbit. At least a portion of the antibody or antibody
fragment
may be from a human. At least a portion of the antibody or antibody fragment
antibody
may be from cynomolgus monkey.
[0244]
The targeting antibody or antibody fragment may be based on or derived
from
an antibody or antibody fragment from a mammal, bird, fish, amphibian or
reptile.
Mammals include, but are not limited to, carnivores, rodents, elephants,
marsupials,
rabbits, bats, primates, seals, anteaters, cetaceans, odd-toed ungulates and
even-toed
ungulates. The mammal may be a human, non-human primate, mouse, sheep, cat,
dog,
cow, horse, goat, or pig.
[0245] The targeting antibody or an antibody fragment may recognise or bind an

antigen selected from, by non-limiting example, CD19, Her2, CLL-1, CD33,
EGFRvIll,
CD20, 0D22, BCMA or a fragment thereof. The antigen may comprise a wild-type
antigen. The antigen may comprise one or more mutations.
[0246] The targeting antibody or antibody fragment may be an anti-CD19
antibody or
a fragment thereof. The targeting polypeptide may be an anti-CD22 antibody.
The
targeting polypeptide may be an anti-BCMA antibody or a fragment thereof. The
targeting polypeptide may be an anti-EGFRvIll antibody or a fragment thereof.
The
targeting polypeptide may be an anti-Her2 antibody or a fragment thereof. The
targeting
polypeptide may comprise an anti-CD20 antibody or antibody fragment. The
targeting
polypeptide may comprise rituximab. The targeting polypeptide may comprise an
anti-
EGFR antibody or antibody fragment. The targeting polypeptide may comprise an
anti-
CEA antibody or antibody fragment. The targeting polypeptide may comprise an
anti-
CLL-1 antibody or antibody fragment. The targeting polypeptide may comprise an
anti-
CD33 antibody or antibody fragment. The targeting polypeptide may comprise an
anti-
EpCAM antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
CD30 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
CD79B antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
CD37 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
CD38 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
CD70 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
CD276 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
GD2 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
CD371 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
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CD135 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
CD105 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
CD123 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
ROR-1 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
PD-L1 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
MET-R antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
PDGFRalpha antibody or fragment thereof. The targeting polypeptide may
comprise an
anti-Her3 antibody or fragment thereof. The targeting polypeptide may comprise
an anti-
FRalpha antibody or fragment thereof. The targeting polypeptide may comprise
an anti-
GPC3 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
SLAMf7 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
TNFRSF1OB antibody or fragment thereof. The targeting polypeptide may comprise
an
anti-GPNMB antibody or fragment thereof. The targeting polypeptide may
comprise an
anti-VEGFR2 antibody or fragment thereof. The targeting polypeptide may
comprise an
anti-a1pha4beta7/ alphaEbeta7 antibody or fragment thereof. The targeting
polypeptide
may comprise an anti-CSPG4 antibody or fragment thereof. The targeting
polypeptide
may comprise an anti-CD80 antibody or fragment thereof. The targeting
polypeptide
may comprise an anti-CCR4 antibody or fragment thereof. The targeting
polypeptide
may comprise an anti-CD115 antibody or fragment thereof. The targeting
polypeptide
may comprise an anti-ENOX-2 antibody or fragment thereof. The targeting
polypeptide
may comprise an anti-CD56 antibody or fragment thereof. The targeting
polypeptide
may comprise an anti-huVH1-69 antibody or fragment thereof. The targeting
polypeptide may comprise an anti-CD117 antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-CD133 antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-MUC1 antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-MSLN antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-ROR-2 antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-IL13Ra2 antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-EPHA2 antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-EGFRvIll antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-PSMA antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-CEA antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-Lewis Y antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-PSCA antibody or fragment thereof. The
targeting
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polypeptide may comprise an anti-MUC1 antibody or fragment thereof. The
targeting
polypeptide may comprise an anti-CD181L1CAM antibody or fragment thereof. The
targeting polypeptide may comprise an anti-EpCAM antibody or fragment thereof.
The
targeting polypeptide may comprise an anti-ALK antibody or fragment thereof.
The
targeting polypeptide may comprise an anti-IGF-1R CD221 antibody or fragment
thereof. The targeting polypeptide may comprise an anti-Nectin 4 antibody or
fragment
thereof. The targeting polypeptide may comprise an anti-FAP antibody or
fragment
thereof. The targeting polypeptide may comprise an anti-AXL antibody or
fragment
thereof. The targeting polypeptide may comprise an anti-CD138 antibody or
fragment
thereof. The targeting polypeptide may comprise an anti-CLDN6 antibody or
fragment
thereof. The targeting polypeptide may comprise an anti-Her4 antibody or
fragment
thereof. The targeting polypeptide may comprise an anti-Claudin 18.2 antibody
or
fragment thereof. The targeting polypeptide may comprise an anti-0-acetylated
GD2
antibody or fragment thereof. The targeting polypeptide may comprise an anti-
GD3
antibody or fragment thereof_ The targeting polypeptide may comprise an anti-
GM2
antibody or fragment thereof. The targeting polypeptide may comprise an anti-
TM4SF1
antibody or fragment thereof. The targeting polypeptide may comprise an anti-
CD147
antibody or fragment thereof. The targeting polypeptide may comprise an anti-
CEACAM5 antibody or fragment thereof. The targeting polypeptide may comprise
an
anti-VEGFR-1 antibody or fragment thereof. The targeting polypeptide may
comprise an
anti-PDPN antibody or fragment thereof. The targeting polypeptide may comprise
an
anti-VVT1 antibody or fragment thereof. The targeting polypeptide may comprise
an anti-
GPC2 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
FGFR4 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
EphB4 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
STEAP-1 antibody or fragment thereof. The targeting polypeptide may comprise
an anti-
STEAP-2 antibody or fragment thereof. The targeting polypeptide may comprise
an anti-
MUC1 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
chlorotoxin antibody or fragment thereof. The targeting polypeptide may
comprise an
anti-206 antibody or fragment thereof. The targeting polypeptide may comprise
an anti-
ILRAP1 antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
MICA antibody or fragment thereof. The targeting polypeptide may comprise an
anti-
MAGE-Al scTcR antibody or fragment thereof. The targeting polypeptide may
comprise
an anti-MAGE-Al sTCR antibody or fragment thereof. The targeting polypeptide
The
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targeting polypeptide may comprise an anti-MICA antibody or fragment thereof.
The
targeting polypeptide may comprise an anti-TRBC1/2 antibody or fragment
thereof. The
targeting polypeptide may comprise an anti-B7-H7 antibody or fragment thereof.
The
targeting polypeptide may comprise an anti-CD34 antibody or fragment thereof.
The
targeting polypeptide may comprise an anti-CD7 antibody or fragment thereof.
The
targeting polypeptide may comprise an anti-TIM3 antibody or fragment thereof.
The
targeting polypeptide may comprise an anti-CD191 antibody or fragment thereof.
The
targeting polypeptide may comprise an anti-CD66b antibody or fragment thereof.
The
targeting polypeptide may comprise an anti-CD11b antibody or fragment thereof.
The
targeting polypeptide may comprise an anti-EMR2 antibody or fragment thereof.
The
targeting polypeptide may comprise an anti-M UC16 antibody or fragment
thereof. The
targeting polypeptide may comprise an anti-NYESO-1 HLA-A2 antibody or fragment

thereof or a soluble TcR or variant thereof. The targeting polypeptide may
comprise an
anti-SURVIVIN HLA-A2 antibody or fragment thereof or a soluble TcR or variant
thereof.
The targeting polypeptide may comprise an anti-CD200 antibody or fragment
thereof.
[0247] The targeting antibody or antibody fragment may be selected from any
commercially available antibody. The targeting antibody or antibody fragment
may be
selected from trastuzumab (for binding to Her2), alemtuzumab (for binding
CD52),
bevacizumab (for binding VEGF-A), brentuximab (for binding 0030, gemtuzumab
(for
binding CD33), ipilimumab (for binding VTLA-4), ibritumomab (for binding
CD20),
panitumumab (for binding EGFR), cetuximab (for binding EGFR), rituximab (for
binding
CD20), and fragments thereof_
[0248] The targeting antibody or antibody fragment may be any referred to in
Table 1,
or an antigen binding fragment at least 80%, at least 81%, at least 82%, at
least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%,
at least 97%, at least 98%, or at least 99% identical thereto.
[0249] The targeting moiety of the bridging molecule may target peptide MHC
complexes and in such embodiments, the target moiety may be a soluble TcR
molecule
or single chain TcR molecule.
[0250] Non-limiting examples of the sequences of various targeting antibodies,
or
antigen binding fragments thereof, are provided herein in Table 1.
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[0251] It will be well within the purview of the skilled person to be able to
determine
the appropriate design of a bridging molecule as described herein, including
selection of
the targeting moiety of the bridging molecule.
[0252] More specifically, the skilled person, having knowledge of the CAR
requiring
"redirection" as described herein would be able to select an alternative
targeting moiety
to which the CAR should be directed, to facilitate treatment of the cancer or
disease
requiring treatment.
[0253] As described herein, the targeting moiety may be for binding to any
cell
surface molecule on a target cell (such as a tumour-associated antigen
expressed on a
cancer cell). The skilled person can select an appropriate cell surface
molecule
depending on the disease (eg cancer) requiring treatment. For example, if the
disease
is a Her2+ cancer, then the skilled person may select a targeting moiety that
is capable
of specifically binding to Her2 on the cancer cell. Similarly, if the disease
is a CD19+
cancer cell, then the skilled person may select a targeting moiety that is
capable of
specifically binding to CD19 on the cancer. The skilled person will be
familiar with
methods for determining the antigen expression profile of the cancer or
condition to be
treated in order to identify suitable surface molecules expressed on the
target cell.
[0254] Having identified a suitable cell surface molecule (target antigen) on
the target
cell (eg: CD19, Her2, CLL-1, 0D33, EGFRvIll, CD20, CD22, BCMA or any other
tumour-associated antigen described herein or that may be selected for
targeting), the
skilled person can readily determine the sequence of a binding domain that can
bind to
said target antigen, including by reference to commercially available
antibodies or to
published literature describing antigen binding domains of known antibodies
that bind to
the antigen. The skilled person can then formulate the structure of a bridging
molecule
(fusion protein) as described herein, comprising a targeting moiety that binds
to the cell
surface molecule on a target cell. The composition of the remaining components
of the
bridging molecule (eg: the tumour-specific antigen epitope moiety) are
described further
herein.
[0255]
Finally, the skilled person, having identified a suitable cell surface
molecule
(target antigen) on the target cell, and tumour-specific antigen epitope
moiety in order to
arrive at a bridging molecule of the invention, can readily determine, using
routine
techniques, whether the bridging molecule: a) binds to the target cell, b)
binds to the
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CAR T cell and c) can successfully redirect the CAR T cell to the target cells
to induce
cell killing. Methods of determining binding to target antigens, and
cytotoxicity are well
known in the art. Non-limiting methods and various experimental protocols for
determining binding to target cell, CAR T cell and induction of cell killing
are described
in detail herein in the Examples.
Dysfunctional P2X7 receptor epitope moiety
[0256] The present invention contemplates the use of a tumour-specific antigen

epitope moiety in the form of an epitope from dysfunctional P2X7 receptor (ie
wherein
the tumour-specific antigen is dysfunctional P2X7 receptor).
[0257] A dysfunctional P2X7 receptor epitope moiety may be provided in the
form of a
dysfunctional P2X7 receptor, or a fragment of a dysfunctional P2X7 receptor,
that has at
least one of the three ATP binding sites that are formed at the interface
between
adjacent correctly packed monomers that are unable to bind ATP. Such receptors
are
unable to extend the opening of the non-selective calcium channels to
apoptotic pores.
[0258] A range of peptide fragments of a dysfunctional P2X7 receptor are known
and
discussed in PCT/AU2002/000061 (and in corresponding publications WO
2002/057306
and US 7,326,415, US 7,888,473, US 7,531,171, US 8,080,635, US 8,399,617, US
8,709,425, US 9,663,584, or US 10,450,380), PCT/AU2008/001364 (and in
corresponding publications WO 2009/033233 and US 8,440,186, US 9,181,320, US
9,944,701 or US 10,597,45) and PCT/AU2009/000869 (and in corresponding
publications WO 2010/000041 and US 8,597,643, US 9,328,155 or US 10,238,716)
the
contents of all of which are incorporated in entirety. Exemplary peptides
within these
specifications that include epitopes contemplated for use in this invention
are described
below.
PC T publication Peptide sequence
WO 2002/057306 GHNYTTRNILPGLNITC (SEQ ID NO: 2) (also
referred to
herein as the "E200" epitope)
WO 2009/033233 KYYKENNVEKRTLIKVF (SEQ ID NO: 12) (also
referred to
herein as the "E300" epitope)
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WO 2010/000041
GHNYTTRNILPGAGAKYYKENNVEK (SEQ ID NO: 14)
(also referred to herein as the "E200/E300" or "composite" epitope)
[0259] In any embodiment, the amino acid sequence of the dysfunctional P2X7
receptor epitope moiety of any bridging molecule described herein, comprises
or
consists of a sequence as set forth in any of SEQ ID Nos: 2 to 30, 168, and
SEQ ID
NOs: 365-400, or sequences at least 80%, at least 81%, at least 82%, at least
83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%,
at least 97%, at least 98%, or at least 99%, identical thereto. Preferably,
the
dysfunctional P2X7 receptor epitope moiety comprises at least the sequence of
SEQ ID
NO: 11.
[0260] The dysfunctional P2X7 receptor epitope moiety may have any functional
chemical group such as a carboxyl group, an active ester, an acetamide or
maleimide
capable of coupling to a targeting moiety as disclosed herein, for example an
antibody
or fragment thereof using NH2 or SH groups for coupling thereto.
[0261] Various examples of dysfunctional P2X7 receptor epitope moieties are
described herein, particularly in Table 1. As is clear from Table 1, the
minimum E200
sequence comprises at least the sequence of SEQ ID NO: 11. In preferred
embodiments, the minimum E200 sequence comprises at least the sequence of SEQ
ID
NO: 2, or a variant thereof, such as the sequence of SEQ ID NO: 4.
[0262] In certain examples, the E200 epitope for inclusion in the bridging
molecules
of the invention, may comprise extensions in the N-terminal and/or C-terminal
region.
The extensions may be derived from the naturally occurring nfP2X7 receptor
sequence,
and may comprise extensions of at least 1 amino acid residue, at least 2 amino
acid
residues, at least 3, at least 4, at least 5 or more amino acid residues. For
example, the
E200 epitope defined in SEQ ID NO: 4 may comprise an N-terminal extension of
the
sequence DFP, which corresponds to the three amino acid residues immediately N

terminal to the E200 sequence in SEQ ID NO: 1. Further, the E200 epitope
defined in
SEQ ID NO: 4 may comprise a C terminal extension, as depicted, for example in
SEQ
ID NO: 6 and 7. It will be appreciated that such N and C terminal extensions
may serve
to improve the efficacy of binding of the CAR-T cell to the epitope on the
bridging
molecule.
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[0263] In any embodiment, the dysfunctional P2X7 receptor epitope moiety is
conjugated to the targeting moiety so as to enable sufficient accessibility
for binding by
an nfP2X7 receptor CAR. Thus, while the minimum dysfunctional P2X7 receptor
epitope
moiety sequence comprises the minimum E200 sequence at least the sequence of
SEQ
ID NO: 11, in preferred embodiments, the minimum E200 sequence comprises at
least
the sequence of SEQ ID NO: 2, or a variant thereof, such as the sequence of
SEQ ID
NO: 4, in addition to one or more linker or hinge regions for enabling binding
by an
nfP2X7 receptor CAR. In certain embodiments, the total length of the
dysfunctional P2X7
receptor epitope moiety (including linker and hinge region) is at least 17
amino acids in
length, or at least, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33,34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45, 47, 48, 49, or 50 amino acids in
length.
Preferably the dysfunctional P2X7 receptor epitope moiety is no more than
about 100
amino acids in length, more preferably, no more than about 99, 98, 97, 96, 95,
94, 93,
92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74,
73, 72, 71, 70,
69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 or
50 amino
acids in length.
[0264] In the context of a bridging molecule for targeting CD19, preferably
the
dysfunctional P2X7 receptor epitope moiety is no more than about 35 amino
acids in
length and at least 20 amino acids in length, preferably at least 21, 22, 23,
24, 25, or 26
amino acids in length.
[0265] In the context of a bridging molecule for targeting CD33, preferably
the
dysfunctional P2X7 receptor epitope moiety is no more than about 45 amino
acids in
length and at least 20 amino acids in length, preferably at least 21, 22, 23,
24, 25, or 26
amino acids in length.
EGFRyll I epitope moiety
[0266] In any embodiment, the amino acid sequence of the EGFRvIll epitope
moiety
of any bridging molecule described herein, comprises or consists of a sequence
as set
forth in any of SEQ ID Nos: 267, or sequences at least 80%, at least 81%, at
least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical
thereto.
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Preferably, the EGFRvIll epitope moiety comprises at least the sequence of SEQ
ID
NO: 267.
CLDN6 epitope moiety
[0267] In any embodiment, the amino acid sequence of the CLDN6 epitope moiety
of
any bridging molecule described herein, comprises or consists of a sequence as
set
forth in any of SEQ ID Nos: 273, 274 or 275, or sequences at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto. Preferably, the CLDN6 epitope moiety comprises at least the sequence
of SEQ
ID NO: 273, 274 or 275.
Exemplary bridging molecules
[0268] The present specification provides various non-limiting examples of
tumour-
specific antigen epitope moiety (e.g. dysfunctional P2X7 receptor epitope
moiety) /
targeting moiety pairs.
[0269] Exemplary bridging molecules of the invention are described in Table 1.
For
those bridging molecules that are described in Table 1 that include a nfP2X7
epitope
moiety, the specification includes those bridges but with the nfP2X7 epitope
moiety
substituted for a EGFRyl II or CLDN6 epitope moiety.
[0270] In examples where the bridging molecules comprise a targeting moiety
for
binding to CD19, the targeting moiety may comprise or consist of a heavy and
paired
light variable chain combination as set forth in SEQ ID NOs: 31 and 32; or 143
and 144
(heavy and light chain, respectively; or sequences at least 80%, at least 81%,
at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0271] In the above examples, the bridging molecules may comprise the tumour-
specific antigen epitope moiety (e.g. dysfunctional P2X7 receptor epitope
moiety)
conjugated to the heavy chain, or the tumour-specific antigen epitope moiety
(e.g.
dysfunctional P2X7 receptor epitope moiety) conjugated to the light chain.
Preferably,
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the tumour-specific antigen epitope moiety (e.g. dysfunctional P2X7 receptor
epitope
moiety) is conjugated to the light chain of the target binding moiety.
[0272] In any embodiment wherein the bridging molecule comprises CD19-binding
heavy/light chain pairs where the heavy chain comprises or consists of the
dysfunctional
P2X7 receptor epitope moiety, the sequences of the variable sequences of the
heavy
and light chain pairs are preferably selected from: SEQ ID NOs: 33 and 32; 34
and 32,
37 and 32; 37 and 38; (heavy and light chain sequences recited, respectively)
or
sequences at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, identical thereto.
[0273] In any embodiment wherein the bridging molecule comprises CD19-binding
heavy/light chain pairs where the light chain comprises or consists of the
dysfunctional
P2X7 receptor epitope moiety, the sequences of the variable sequences of the
heavy
and light chain pairs are preferably selected from: SEQ ID NOs: 31 and 35; 31
and 36;
39 and 31; 52 and 51; 143 and 145; 143 and 146; 143 and 147; 143 and 148; 143
and
149; 143 and 150; 143 and 151; 143 and 152; 143 and 153; 143 and 154; 143 and
155;
143 and 156; 143 and 157; 143 and 158; 143 and 159; 143 and 160; 143 and 161;
143
and 162; 143 and 163; or 143 and 164 (heavy and light chain sequences recited,

respectively) or sequences at least 80%, at least 81%, at least 82%, at least
83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%,
at least 97%, at least 98%, or at least 99%, identical thereto. In another
embodiment
wherein the bridging molecule comprises CD19-binding heavy/light chain pairs
where
the light chain is any one of the light chains above and a heavy chain
selected from
SEQ ID NO: 141 or 142.
[0274] The targeting moiety may be in the form of an scFv comprising a heavy
and a
light chain.
[0275] In any embodiment, a CD19-binding scFv for use in the bridging
molecules of
the invention may be one having a sequence as set forth in SEQ ID NOs: 40 or
41 or
sequences at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%,
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at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, identical thereto. It will be appreciated that in
the context of
an scFv, the dysfunctional P2X7 receptor epitope moiety may be conjugated to
the light
chain of the scFv, such as in any of SEQ ID NOs: 42, 43, 46, 48, or to the
heavy chain
of the scFv, such as in any of SEQ ID NOs: 44, 45, 47, 49, 50 or sequences at
least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, or at
least 99%, identical thereto.
[0276] In preferred embodiments, the bridging molecule for binding to 0019
comprises a targeting moiety comprising an antigen binding domain for
specifically
binding to CD19, preferably as described herein (more preferably, comprising
the amino
acid sequence as set forth in SEQ ID NOs: 52/143 or 143/144 (heavy and light
chains
respectively) or a sequence at least 80%, at least 81%, at least 82%, at least
83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%,
at least 97%, at least 98%, or at least 99%, identical thereto) and comprises
a tumour-
specific antigen epitope moiety comprising or consisting of the amino acid
sequence set
forth in any of SEQ ID NOs: 365, 371, 377, 383, 389, and 395.
[0277] In any embodiment, a bridging molecule for binding to CD20 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 53 and 54, or in 55 and 56
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0278] In any embodiment, a bridging molecule for binding to CO22 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 57 and 58; or in 59 and 60
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
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[0279] In any embodiment, a bridging molecule for binding to CD79B may
comprise
or consist of the sequences set forth in SEQ ID NOs: 61 and 62, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0280] In any embodiment, a bridging molecule for binding to CD37 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 63 and 64, (light and heavy
chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0281] In any embodiment, a bridging molecule for binding to CD38 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 65 and 66, (light and heavy
chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0282] In any embodiment, a bridging molecule for binding to CD70 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 67 and 68, (light and heavy
chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0283] In any embodiment, a bridging molecule for binding to CD30 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 39 and 70, (light and heavy
chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
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[0284] In any embodiment, a bridging molecule for binding to CD33 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 71 and 72 or 73 and 74,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0285] In preferred embodiments, the bridging molecule for binding to 0033
comprises a targeting moiety comprising an antigen binding domain for
specifically
binding to 0D33, preferably as described herein (more preferably, comprising
the amino
acid sequence as set forth in SEQ ID NOs: 73 and 72 or 74, (light and heavy
chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto),
and comprises a tumour-specific antigen epitope moiety comprising or
consisting of the
amino acid sequence set forth in any of SEQ ID NOs: 365, 373, 377, 388, 390,
and 395.
[0286] In any embodiment, a bridging molecule for binding to Her2 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 75 and 75; or 77 and 78,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0287] In any embodiment, a bridging molecule for binding to EGFR may comprise
or
consist of the sequences set forth in SEQ ID NOs: 79 and 80 or 81 and 82 or 83
and 84,
(light and heavy chain sequences recited, respectively) or a sequence at least
80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99%,
identical thereto.
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[0288] In any embodiment, a bridging molecule for binding to CD276 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 85 and 86, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0289] In any embodiment, a bridging molecule for binding to GD2 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 87 and 88, (light and heavy
chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0290] In any embodiment, a bridging molecule for binding to BCMA may comprise
or
consist of the sequences set forth in SEQ ID NOs: 89 and 90, (light and heavy
chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0291] In any embodiment, a bridging molecule for binding to CD371 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 91 and 92, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0292] In any embodiment, a bridging molecule for binding to CD135 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 93 and 94, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
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[0293] In any embodiment, a bridging molecule for binding to CD123 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 95 and 95, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0294] In any embodiment, a bridging molecule for binding to CD105 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 97 and 98, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0295] In any embodiment, a bridging molecule for binding to ROR-1 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 99 and 100, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0296] In any embodiment, a bridging molecule for binding to PD-L1 may
comprise or
consist of the sequences set forth in SEQ ID NOs: 101 and 102, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0297] In any embodiment, a bridging molecule for binding to MET-R may
comprise
or consist of the sequences set forth in SEQ ID NOs: 103 ad 104, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
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[0298] In any embodiment, a bridging molecule for binding to PDGFRalpha may
comprise or consist of the sequences set forth in SEQ ID NOs: 105 and 106 or
107 and
108 (light and heavy chain sequences recited, respectively) or a sequence at
least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least
99%, identical thereto.
[0299] In any embodiment, a bridging molecule for binding to Her3 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 109 and 110, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0300] In any embodiment, a bridging molecule for binding to FRalpha may
comprise
or consist of the sequences set forth in SEQ ID NOs: 111 and 112, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0301] In any embodiment, a bridging molecule for binding to CGPC3 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 113 and 114, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0302] In any embodiment, a bridging molecule for binding to SLAMF7 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 115 and 116, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
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[0303] In any embodiment, a bridging molecule for binding to TNFRSF1OB may
comprise or consist of the sequences set forth in SEQ ID NOs: 117 and 118,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0304] In any embodiment, a bridging molecule for binding to GPNMB may
comprise
or consist of the sequences set forth in SEQ ID NOs: 119 and 120, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0305] In any embodiment, a bridging molecule for binding to VEGFR2 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 121 and 122, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0306] In any embodiment, a bridging molecule for binding to a4137 and/or
aE137 may
comprise or consist of the sequences set forth in SEQ ID NOs: 123 and 124; or
125 and
126, (light and heavy chain sequences recited, respectively) or a sequence at
least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, or at
least 99%, identical thereto.
[0307] In any embodiment, a bridging molecule for binding to CSPG4 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 127 and 128, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
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[0308] In any embodiment, a bridging molecule for binding to CD80 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 129 and 130, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0309] In any embodiment, a bridging molecule for binding to CCR4 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 131 and 132, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0310] In any embodiment, a bridging molecule for binding to CD115 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 133 and 134, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0311] In any embodiment, a bridging molecule for binding to ENOX-2 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 135 and 136, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0312] In any embodiment, a bridging molecule for binding to CD56 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 137 and 138, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
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[0313] In any embodiment, a bridging molecule for binding to huVH1-69 may
comprise or consist of the sequences set forth in SEQ ID NOs: 139 and 140,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0314] In any embodiment, a bridging molecule for binding to CD117 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 169 and 170, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0315] In any embodiment, a bridging molecule for binding to CD133 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 171 and 172, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0316] In any embodiment, a bridging molecule for binding to MUC1 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 173 and 174, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0317] In any embodiment, a bridging molecule for binding to mesothelin may
comprise or consist of the sequences set forth in SEQ ID NOs: 175 and 176,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
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[0318] In any embodiment, a bridging molecule for binding to ROR2 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 177 and 178, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0319] In any embodiment, a bridging molecule for binding to I L13Ra2 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 179 and 180, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0320] In any embodiment, a bridging molecule for binding to I L13Ra2 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 181, or a sequence at
least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least
99%, identical thereto.
[0321] In any embodiment, a bridging molecule for binding to EPHA2 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 182 and 183, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0322] In any embodiment, a bridging molecule for binding to EGFRvIll may
comprise
or consist of the sequences set forth in SEQ ID NOs: 184 and 185, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
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[0323] In any embodiment, a bridging molecule for binding to PSMA may comprise
or
consist of the sequences set forth in SEQ ID NOs: 186 and 187, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0324] In any embodiment, a bridging molecule for binding to CEA may comprise
or
consist of the sequences set forth in SEQ ID NOs: 188 and 189, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0325] In any embodiment, a bridging molecule for binding to PSCA may comprise
or
consist of the sequences set forth in SEQ ID NOs: 190 and 191, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0326] In any embodiment, a bridging molecule for binding to Lewis Y may
comprise
or consist of the sequences set forth in SEQ ID NOs: 192 and 193, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0327] In any embodiment, a bridging molecule for binding to CD171 L1CAM may
comprise or consist of the sequences set forth in SEQ ID NOs: 194 and 195,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
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[0328] In any embodiment, a bridging molecule for binding to EpCAM may
comprise
or consist of the sequences set forth in SEQ ID NOs: 196 and 197, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0329] In any embodiment, a bridging molecule for binding to ALK may comprise
or
consist of the sequences set forth in SEQ ID NOs: 198 and 199, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0330] In any embodiment, a bridging molecule for binding to IGF-1R CD221 may
comprise or consist of the sequences set forth in SEQ ID NOs: 200 and 201,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0331] In any embodiment, a bridging molecule for binding to Nectin 4 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 202 and 203, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0332] In any embodiment, a bridging molecule for binding to FAP may comprise
or
consist of the sequences set forth in SEQ ID NOs: 204 and 205, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
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[0333] In any embodiment, a bridging molecule for binding to AXL may comprise
or
consist of the sequences set forth in SEQ ID NOs: 206 and 207, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0334] In any embodiment, a bridging molecule for binding to CD138 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 208 and 209, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0335] In any embodiment, a bridging molecule for binding to CLDN6 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 210 and 211, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0336] In any embodiment, a bridging molecule for binding to Her4 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 212 and 213, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0337] In any embodiment, a bridging molecule for binding to Claudin 18.2 may
comprise or consist of the sequences set forth in SEQ ID NOs: 214 and 215,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
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[0338] In any embodiment, a bridging molecule for binding to 0-acetylated GD2
may
comprise or consist of the sequences set forth in SEQ ID NOs: 216 and 217,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0339] In any embodiment, a bridging molecule for binding to GD3 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 218 and 219, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0340] In any embodiment, a bridging molecule for binding to GM2 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 220 and 221, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0341] In any embodiment, a bridging molecule for binding to TM4SF1 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 222 and 223, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0342] In any embodiment, a bridging molecule for binding to CD147 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 224 and 225, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
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[0343] In any embodiment, a bridging molecule for binding to CEACAM5 may
comprise or consist of the sequences set forth in SEQ ID NOs: 226 and 227,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0344] In any embodiment, a bridging molecule for binding to VEGFR-1 may
comprise or consist of the sequences set forth in SEQ ID NOs: 228 and 229,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0345] In any embodiment, a bridging molecule for binding to Podoplanin (PDPN)

may comprise or consist of the sequences set forth in SEQ ID NOs: 230 and 231,
(light
and heavy chain sequences recited, respectively) or a sequence at least 80%,
at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at
least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
identical thereto.
[0346] In any embodiment, a bridging molecule for binding to \A/T1 may
comprise or
consist of the sequences set forth in SEQ ID NOs: 232 and 233, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0347] In any embodiment, a bridging molecule for binding to GPC2 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 234 and 235, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
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at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0348] In any embodiment, a bridging molecule for binding to FGFR4 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 236 and 237, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0349] In any embodiment, a bridging molecule for binding to EphB4 may
comprise or
consist of the sequences set forth in SEQ ID NOs: 238 and 239, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0350] In any embodiment, a bridging molecule for binding to STEAP-1 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 240 and 241, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0351] In any embodiment, a bridging molecule for binding to STEAP-2 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 242 and 243, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0352] In any embodiment, a bridging molecule for binding to IL11Ra may
comprise
or consist of the sequences set forth in SEQ ID NOs: 244 and 245, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
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88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0353] In any embodiment, a bridging molecule for binding to CD163 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 246 and 247, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0354] In any embodiment, a bridging molecule for binding to Chlorotoxin may
comprise or consist of the sequences set forth in SEQ ID NOs: 248 and 249,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0355] In any embodiment, a bridging molecule for binding to CD206 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 250, (heavy chain
sequence
recited) or a sequence at least 80%, at least 81%, at least 82%, at least 83%,
at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at
least 97%, at least 98%, or at least 99%, identical thereto.
[0356] In any embodiment, a bridging molecule for binding to IL1RAP may
comprise
or consist of the sequences set forth in SEQ ID NOs: 251 and 252, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0357] In any embodiment, a bridging molecule for binding to MICA may comprise
or
consist of the sequences set forth in SEQ ID NOs: 253 and 254, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
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at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0358] In any embodiment, a bridging molecule for binding to MAGE-Al may
comprise or consist of the sequences set forth in SEQ ID NOs: 255, or a
sequence at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least
86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, or at
least 99%, identical thereto.
[0359] In any embodiment, a bridging molecule for binding to MAGE-Al may
comprise or consist of the sequences set forth in SEQ ID NOs: 256 and 257, or
a
sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, identical thereto.
[0360] In any embodiment, a bridging molecule for binding to MAGE-Al may
comprise or consist of the sequences set forth in SEQ ID NOs: 258 and 259, or
a
sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, identical thereto.
[0361] In any embodiment, a bridging molecule for binding to TRBC1 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 260 and 261, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0362] In any embodiment, a bridging molecule for binding to TRBC2 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 262 and 263, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
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88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0363] In any embodiment, a bridging molecule for binding to urokinase-type
plasminogen activator receptor (uPAR) may comprise or consist of the sequences
set
forth in SEQ ID NOs: 264 and 265, (light and heavy chain sequences recited,
respectively) or a sequence at least 80%, at least 81%, at least 82%, at least
83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%,
at least 97%, at least 98%, or at least 99%, identical thereto.
[0364] In any embodiment, a bridging molecule for binding to CD33 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 268 and 269, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0365] In any embodiment, a bridging molecule for binding to Her2 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 276 and 277, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0366] In any embodiment, a bridging molecule for binding to CD33 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 278 and 279, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0367] In any embodiment, a bridging molecule for binding to Her2 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 270 and 271, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
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at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0368] In any embodiment, a bridging molecule for binding to B7-H7 (HHLA2) may

comprise or consist of the sequences set forth in SEQ ID NOs: 280 and 281,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0369] In any embodiment, a bridging molecule for binding to CD34 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 282 and 283, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0370] In any embodiment, a bridging molecule for binding to CD7 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 284 and 285, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0371] In any embodiment, a bridging molecule for binding to CD7 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 286, (heavy chain sequence)
or a
sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, identical thereto.
[0372] In any embodiment, a bridging molecule for binding to GPRC5D may
comprise
or consist of the sequences set forth in SEQ ID NOs: 287 and 288, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
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88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0373] In any embodiment, a bridging molecule for binding to TIM-3 may
comprise or
consist of the sequences set forth in SEQ ID NOs: 289 and 290, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0374] In any embodiment, a bridging molecule for binding to 00191 (CCR1) may
comprise or consist of the sequences set forth in SEQ ID NOs: 291 and 292,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0375] In any embodiment, a bridging molecule for binding to CD66b (CEACAM8)
may comprise or consist of the sequences set forth in SEQ ID NOs: 293 and 294,
(light
and heavy chain sequences recited, respectively) or a sequence at least 80%,
at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at
least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%,
identical thereto.
[0376] In any embodiment, a bridging molecule for binding to CD11b (MAC-1) may

comprise or consist of the sequences set forth in SEQ ID NOs: 295 and 296,
(light and
heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0377] In any embodiment, a bridging molecule for binding to EMR2 (ADGRE2) may

comprise or consist of the sequences set forth in SEQ ID NOs: 297 and 298,
(light and
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heavy chain sequences recited, respectively) or a sequence at least 80%, at
least 81%,
at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical
thereto.
[0378] In any embodiment, a bridging molecule for binding to MUC16 may
comprise
or consist of the sequences set forth in SEQ ID NOs: 299 and 300, (light and
heavy
chain sequences recited, respectively) or a sequence at least 80%, at least
81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0379] In any embodiment, a bridging molecule for binding to NYESO-1 HLA-A2
may
comprise or consist of the sequences set forth in SEQ ID NOs: 301 and 302, or
a
sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, identical thereto.
[0380] In any embodiment, a bridging molecule for binding to Survivin HLA-A2
may
comprise or consist of the sequences set forth in SEQ ID NOs: 303 and 304, or
a
sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at
least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at
least 98%, or at least 99%, identical thereto.
[0381] In any embodiment, a bridging molecule for binding to BCMA may comprise
or
consist of the sequences set forth in SEQ ID NOs: 305, or a sequence at least
80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99%,
identical thereto.
[0382] In any embodiment, a bridging molecule for binding to BCMA may comprise
or
consist of the sequences set forth in SEQ ID NOs: 306, or a sequence at least
80%, at
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least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least
86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99%,
identical thereto.
[0383] In any embodiment, a bridging molecule for binding to C200 may comprise
or
consist of the sequences set forth in SEQ ID NOs: 308 and 307, (light and
heavy chain
sequences recited, respectively) or a sequence at least 80%, at least 81%, at
least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,
identical thereto.
[0384] In any aspect, the bridging molecule described herein does not have a
HIS tag.
Also contemplated, is a bridging molecule that comprises an amino acid
sequence
specified in the Sequence information table above, but without a HIS tag
specified in the
sequence, or a sequence at least 80%, at least 81%, at least 82%, at least
83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at
least 97%, at least 98%, or at least 99%, identical thereto. Further, in one
embodiment,
the bridging molecule may comprise a tag other than a HIS tag, or may comprise
an
amino acid sequence specified in the Sequence information table above but with
a
different tag in the position of the HIS tag specified in the sequence.
Nucleic acids
[0385] In a second aspect, the present invention provides a nucleic acid
molecule
encoding a bridging molecule of the invention, or part thereof.
[0386] The nucleic acid molecule may comprise any polyribonucleotide or
polydeoxyribonucleotide, which may be unmodified, or modified, RNA or DNA. For

example, the nucleic acid molecule may include single- and/or double-stranded
DNA,
DNA that is a mixture of single- and double-stranded regions, single- and
double-
stranded RNA, and RNA that is mixture of single- and double-stranded regions,
hybrid
molecules comprising DNA and RNA that may be single-stranded or, more
typically,
double-stranded or a mixture of single- and double-stranded regions. In
addition, the
nucleic acid molecule may comprise triple-stranded regions comprising RNA or
DNA or
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both RNA and DNA. The nucleic acid molecule may also comprise one or more
modified bases or DNA or RNA backbones modified for stability or for other
reasons. A
variety of modifications can be made to DNA and RNA; thus the term "nucleic
acid
molecule" embraces chemically, enzymatically, or metabolically modified forms.
[0387] In some embodiments of the second aspect of the invention, the nucleic
acid
molecule comprises a nucleic acid sequence encoding the amino acid sequence of
any
one of SEQ ID NOs: 2 to 308. Preferably, the nucleic acid comprises a
nucleotide
sequence encoding the heavy chain and light chain pairs described above.
[0388] Further, the present invention provides a nucleic acid construct
including a
nucleic acid molecule encoding a bridging molecule of the invention, or part
thereof. The
nucleic acid construct may further comprise one or more of: an origin of
replication for
one or more hosts; a selectable marker gene that is active in one or more
hosts; and/or
one or more transcriptional control sequences.
[0389] As used herein, the term "selectable marker gene" includes any gene
that
confers a phenotype on a cell in which it is expressed, to facilitate the
identification
and/or selection of cells that are transfected or transformed with the
construct.
[0390] "Selectable marker genes" include any nucleotide sequences which, when
expressed by a cell transformed with the construct, confer a phenotype on the
cell that
facilitates the identification and/or selection of these transformed cells. A
range of
nucleotide sequences encoding suitable selectable markers are known in the art
(for
example Mortesen, RM. and Kingston RE. Curr Protoc Mol Biol, 2009; Unit 9.5).
Exemplary nucleotide sequences that encode selectable markers include:
Adenosine
deaminase (ADA) gene; Cytosine deaminase (CDA) gene; Dihydrofolate reductase
(DH FR) gene; Histidinol dehydrogenase (hisD) gene; Puromycin-N-acetyl
transferase
(PAC) gene; Thymidine kinase (TK) gene; Xanthine-guanine
phosphoribosyltransferase
(XGPRT) gene or antibiotic resistance genes such as ampicillin-resistance
genes,
puromycin-resistance genes, Bleomycin-resistance genes, hygromycin-resistance
genes, kanamycin-resistance genes and ampicillin-resistance genes; fluorescent

reporter genes such as the green, red, yellow or blue fluorescent protein-
encoding
genes; and luminescence-based reporter genes such as the luciferase gene,
amongst
others which permit optical selection of cells using techniques such as
Fluorescence-
Activated Cell Sorting (FACS).
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[0391] Furthermore, it should be noted that the selectable marker gene may be
a
distinct open reading frame in the construct or may be expressed as a fusion
protein
with another polypeptide (e.g. the CAR).
[0392] As set out above, the nucleic acid construct may also comprise one or
more
transcriptional control sequences. The term "transcriptional control sequence"
should be
understood to include any nucleic acid sequence that effects the transcription
of an
operably connected nucleic acid. A transcriptional control sequence may
include, for
example, a leader, polyadenylation sequence, promoter, enhancer or upstream
activating sequence, and transcription terminator. Typically, a
transcriptional control
sequence at least includes a promoter. The term "promoter" as used herein,
describes
any nucleic acid that confers, activates or enhances expression of a nucleic
acid in a
cell.
[0393] In some embodiments, at least one transcriptional control sequence is
operably connected to the nucleic acid molecule of the second aspect of the
invention.
For the purposes of the present specification, a transcriptional control
sequence is
regarded as "operably connected" to a given nucleic acid molecule when the
transcriptional control sequence is able to promote, inhibit or otherwise
modulate the
transcription of the nucleic acid molecule. Therefore, in some embodiments,
the nucleic
acid molecule is under the control of a transcription control sequence, such
as a
constitutive promoter or an inducible promoter.
[0394] The "nucleic acid construct" may be in any suitable form, such as in
the form
of a plasmid, phage, transposon, cosmid, chromosome, vector, etc., which is
capable of
replication when associated with the proper control elements and which can
transfer
gene sequences, contained within the construct, between cells. Thus, the term
includes
cloning and expression vehicles, as well as viral vectors. In some
embodiments, the
nucleic acid construct is a vector. In some embodiments the vector is a viral
vector.
[0395] A promoter may regulate the expression of an operably connected nucleic
acid
molecule constitutively, or differentially, with respect to the cell, tissue,
or organ at which
expression occurs. As such, the promoter may include, for example, a
constitutive
promoter, or an inducible promoter. A "constitutive promoter" is a promoter
that is active
under most environmental and physiological conditions. An "inducible promoter"
is a
promoter that is active under specific environmental or physiological
conditions. The
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present invention contemplates the use of any promoter that is active in a
cell of
interest. As such, a wide array of promoters would be readily ascertained by
one of
ordinary skill in the art.
[0396] Mammalian constitutive promoters may include, but are not limited to,
Simian
virus 40 (SV40), cytomegalovirus (CMV), P-actin, Ubiquitin C (UBC), elongation
factor-1
alpha (EF1A), phosphoglycerate kinase (PGK) and CMV early enhancer/chicken 13
actin
(CAGG).
[0397] Inducible promoters may include, but are not limited to, chemically
inducible
promoters and physically inducible promoters. Chemically inducible promoters
include
promoters that have activity that is regulated by chemical compounds such as
alcohols,
antibiotics, steroids, metal ions or other compounds. Examples of chemically
inducible
promoters include: tetracycline regulated promoters (e.g. see US Patent
5,851,796 and
US Patent 5,464,758); steroid responsive promoters such as glucocorticoid
receptor
promoters (e.g. see US Patent 5,512,483), ecdysone receptor promoters (e.g.
see US
Patent 6,379,945) and the like; and metal-responsive promoters such as
metallothionein
promoters (e.g. see US Patent 4,940,661, US Patent 4,579,821 and US 4,601,978)

amongst others.
[0398] In the context of the present invention, it will be appreciated that it
may be
desirable in certain circumstances for the expression of the bridging molecule
to be
under the control of an inducible promoter. This enables a switching on and
switching
off of the expression of the nucleic acid encoding the bridging molecule.
[0399] In certain embodiment, and in the case of an inducible expression
construct,
an immune cell expressing a CAR can be genetically modified with a) a nucleic
acid
encoding an antigen binding receptor and b) an inducible expression construct
encoding
the bridging molecule. Upon binding of dysfunctional P2X7 receptor, the immune
cell
induces expression of the gene encoding the bridging molecule. In certain
embodiments, expression of such gene facilitates and/or improves treatment of
cancer.
[0400] As mentioned above, the control sequences may also include a
terminator.
The term "terminator" refers to a DNA sequence at the end of a transcriptional
unit that
signals termination of transcription. Terminators are 3'-non-translated DNA
sequences
generally containing a polyadenylation signal, which facilitate the addition
of
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polyadenylate sequences to the 3'-end of a primary transcript. As with
promoter
sequences, the terminator may be any terminator sequence that is operable in
the cells,
tissues or organs in which it is intended to be used. Suitable terminators
would be
known to a person skilled in the art.
[0401] As will be understood, the nucleic acid constructs of the invention can
further
include additional sequences, for example sequences that permit enhanced
expression,
cytoplasmic or membrane transportation, and location signals. Specific non-
limiting
examples include an Internal Ribosome Entry Site (IRES) or cleavage site (e.g.
P2A,
T2A).
[0402] The present invention extends to all genetic constructs essentially as
described herein. These constructs may further include nucleotide sequences
intended
for the maintenance and/or replication of the genetic construct in eukaryotes
and/or the
integration of the genetic construct or a part thereof into the genome of a
eukaryotic cell.
[0403] Methods are known in the art for the deliberate introduction
(transfection/transduction) of exogenous genetic material, such as the nucleic
acid
construct of the third aspect of the present invention, into eukaryotic cells.
As will be
understood, the method best suited for introducing the nucleic acid construct
into the
desired host cell is dependent on many factors, such as the size of the
nucleic acid
construct, the type of host cell, the desired rate of efficiency of the
transfection/transduction and the final desired, or required, viability of the

transfected/transduced cells. Non-limiting examples of such methods include;
chemical
transfection with chemicals such as cationic polymers, calcium phosphate, or
structures
such as liposonnes and dendrinners; non-chemical methods such as
electroporation,
sonoporation, heat-shock or optical transfection; particle-based methods such
as 'gene
gun' delivery, magnetofection, or impalefection or viral transduction.
[0404] The nucleic acid construct will be selected depending on the desired
method
of transfection/transduction. In some embodiments of the third aspect of the
invention,
the nucleic acid construct is a viral vector, and the method for introducing
the nucleic
acid construct into a host cell is viral transduction. Methods are known in
the art for
utilising viral transduction to elicit expression of a CAR in a PBMC (Parker,
LL. et al.
Hum Gene Ther. 2000;11: 2377-87) and more generally utilising retroviral
systems for
transduction of mammalian cells (Cepko, C. and Pear, W. Curr Protoc Mol Biol.
2001,
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unit 9.9). In other embodiments, the nucleic acid construct is a plasmid, a
cosmid, an
artificial chromosome or the like, and can be transfected into the cell by any
suitable
method known in the art.
Modified cells
[0405] As described herein, the invention includes the use of a cell
expressing a
chimeric antigen receptor comprising an antigen-recognition domain, wherein
the
antigen-recognition domain recognises a tumour-specific antigen (such as
dysfunctional
P2X7 receptor) expressed on a cell surface. The cell may be an "engineered
cell",
"genetically modified cell", "immune cell" or "immune effector cell" as
described herein.
Further, the cell may be capable of differentiating into an immune cell. A
cell that is
capable of differentiating into an immune cell (e.g. T cell that will express
the
dysfunctional P2X7 CAR) may be a stem cell, multi-lineage progenitor cell or
induced
pluripotent stem.
[0406] In any embodiment, the cell may be a T cell, wherein optionally said T
cell
does not express TcRap, PD1, CD3 or CD96 (e.g. by way of knocking down or
knocking out one of these genes on a genetic level or functional level).
[0407] In any embodiment, the cell may be an immune cell, wherein optionally
said
cell does not express accessory molecules that can be checkpoint, exhaustion
or
apoptosis-associated signalling receptors as well as ligands such as PD-1, LAG-
3,
TIGIT, CTLA-4, FAS-L and FAS-R, (e.g. by way of knocking out one of these
genes on
a genetic level or functional level).
[0408] In some embodiments, the genetically modified cell includes two or more

different CARs.
[0409] In some embodiments of the invention, the genetically modified cell
includes a
nucleic acid molecule, or a nucleic acid construct, that encodes for two or
more different
CARs. In some embodiments of the invention, the genetically modified cell
includes two
or more nucleic acid molecules, or two or more nucleic acid constructs, each
of which
encodes for a different CAR.
[0410] As referred to herein, a "genetically modified cell" includes any cell
comprising
a non-naturally occurring and/or introduced nucleic acid molecule or nucleic
acid
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construct encompassed by the present invention. The introduced nucleic acid
molecule
or nucleic acid construct may be maintained in the cell as a discreet DNA
molecule, or it
may be integrated into the genomic DNA of the cell.
[0411] Genomic DNA of a cell should be understood in its broadest context to
include
any and all endogenous DNA that makes up the genetic complement of a cell. As
such,
the genomic DNA of a cell should be understood to include chromosomes,
mitochondria! DNA and the like. As such, the term "genomically integrated"
contemplates chromosomal integration, mitochondria! DNA integration, and the
like. The
"genomically integrated form" of the construct may be all or part of the
construct.
However, in some embodiments the genomically integrated form of the construct
at
least includes the nucleic acid molecule of the second aspect of the
invention.
[0412] As used herein, the term "different CARs" or "different chimeric
antigen
receptors" refers to any two or more CARs that have either non-identical
antigen-
recognition and/or non-identical signalling domains. In one example,
"different CARs"
includes two CARs with the same antigen-recognition domains (e.g. both CARs
may
recognise a dysfunctional P2X7 receptor), but have different signalling
domains, such as
one CAR having a signalling domain with a portion of an activation receptor
and the
other CAR having a signalling domain with a portion of an co-stimulatory
receptor. As
will be understood, at least one of the two or more CARs within this
embodiment will
have an antigen-recognition domain that recognises the dysfunctional P2X7
receptor
and the other CAR(s) may take any suitable form and may be directed against
any
suitable antigen.
[0413] Accordingly, in some embodiments of the invention the two or more
different
CARs have different signalling domains, and may have identical, or different,
antigen-
recognition domains. Specifically, the genetically modified cell of the
invention may
include a first chimeric antigen receptor with a signalling domain that
includes a portion
derived from an activation receptor and a second chimeric antigen receptor
with a
signalling domain including a portion derived from a co-stimulatory receptor.
[0414] In some embodiments, the activation receptor (from which a portion of
signalling domain is derived) is the CD3 co-receptor complex or is an Fc
receptor.
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[0415] In some embodiments, the co-stimulatory receptor (from which a portion
of
signalling domain is derived) is selected from the group consisting of CD27,
CD28, CD-
30, CD40, DAP10, 0X40, 4-1BB (CD137) and !COS.
[0416] In some embodiments, the co-stimulatory receptor (from which a portion
of
signalling domain is derived) is selected from the group consisting of CD28,
0X40 or 4-
1BB.
[0417] In some embodiments, the genetically modified cell is further modified
to
constitutively express co-stimulatory receptors.
[0418] As described above, a cellular immune response is typically only
induced
when an activation signal (typically in response to an antigen) and a co-
stimulation
signal are simultaneously experienced. Therefore, by having a genetically
modified cell
in accordance with some of the above embodiments, which includes two or more
CARs
that in combination provide both an intracellular activation signal and an
intracellular co-
stimulation signal, ensures that a sufficient immune response can be induce in
response
to the recognition by the CAR(s) of their cognate antigen. Alternatively, the
genetically
modified cell may include only one CAR, which has an antigen-recognition
domain that
recognises a dysfunctional P2X7 receptor, and may constitutively express co-
stimulatory
receptors, thereby increasing the likelihood of co-stimulation being provided
simultaneously when the CAR is activated. Alternatively, the genetically
modified cell
may be further modified to constitutively express both co-stimulatory
receptor(s) and
its/their ligand(s). In this way the cell is continuously experiencing co-
stimulation and
only needs the activation of a CAR, with a signalling domain including a
portion from an
activation receptor, for immune activation of the cell.
[0419] Therefore in some embodiments, the genetically modified cell expressing
the
CAR is further modified so as to constitutively express co-stimulatory
receptors. In
further embodiments, the genetically modified cell is further modified so as
to express
ligands for the co-stimulatory receptors, thereby facilitating auto-
stimulation of the cell.
Examples of CAR-expressing T cells that also express both co-stimulatory
receptors
and their cognate ligands (so as to induce auto-stimulation) are known in the
art and
include, inter alia, those disclosed in Stephen MT. et a/. Nat Med, 2007; 13:
1440-9.
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[0420] The potency of a genetically modified cell including a CAR can be
enhanced
by further modifying the cell so as to secrete cytokines, preferably pro-
inflammatory or
pro-proliferative cytokines. This secretion of cytokines provide both
autocrine support for
the cell expressing the CAR, and alters the local environment surrounding the
CAR-
expressing cell such that other cells of the immune system are recruited and
activated.
Consequently, in some embodiments of the fourth or fifth aspects of the
invention the
genetically modified cell is further modified to secret cytokines. This
secretion may be
constitutive, or may be inducible upon recognition of a CAR of its cognate
antigen of
ligand.
[0421] Whilst any one or more cytokines can be selected depending on the
desired
immune response, preferable cytokines and/or chemokines include IL-2, IL-7, IL-
12, IL-
15, IL-17, IL-18 and IL-21, CCL19, CCL21 or a combination thereof.
[0422] The immune cell of the invention can be any suitable immune cell, or
progenitor cell thereof, or can be a homogeneous or a heterogeneous cell
population. In
some embodiments, the cell is a leukocyte, a Peripheral Blood Mononuclear Cell

(PBMC), a lymphocyte, a T cell, a CD4+ T cell, a CD8+ T cell, a natural killer
cell, a
natural killer T cell, or a yo T cell.
[0423] The immune cell may be a T cell, wherein optionally said T cell does
not
express TcRa13, PD1, CD3 or CD96 (e.g. by way of knocking down or knocking out
one
of these genes on a genetic level or functional level).
[0424] The immune cell may not express accessory molecules that can be
checkpoint, exhaustion or apoptosis-associated signalling receptors as well as
ligands
such as PD-1, LAG-3, TIGIT, CTLA-4, FAS-L and FAS-R, (e.g. by way of knocking
out,
or knocking down, one of these genes on a genetic level or functional level).
Methods of treatment and administration
[0425] As discussed further in this document, the present invention finds
application
in the treatment of a variety of conditions, although preferably in the
treatment of
cancers.
[0426] The present invention also contemplates various scenarios for the use
of the
two components of the therapeutics described herein.
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[0427] In one scenario, the individual requiring treatment is administered a
single
composition comprising both the CAR T cells and the bridging molecule.
[0428] In further scenarios, the individual requiring treatment is
administered a
population of CAR T cells, which cells comprise an expression vector encoding
the
bridging molecule. The expression vector may facilitate constitutive or
inducible
expression of the nucleic acid sequence encoding the bridging molecule.
[0429] Further still, the individual requiring treatment may be administered
the CAR T
cells, and at a later date, be administered a composition comprising the
bridging
molecule (e.g., via infusion), or a nucleic acid sequence encoding the
bridging molecule.
Such a scenario may be appropriate in circumstances where the individual is
first
treated with the CAR T cells for targeted treatment of cancers that are
positive for
dysfunctional P2X7 receptor and wherein the subsequent administration of the
bridging
molecule is for the purposes of redirecting the CARs to alternative cancer
antigens, or
to peptides derived from an infectious agent and which are presented on MHC I
or II
molecules of cells.
[0430] Thus the bridging molecule may be administered prior to, at the same
time as,
or after the subject receives treatment with the CAR T cell.
[0431] Where the bridging molecule and CAR T cells are administered to the
subject
at the same time, they can be administered via the same route of
administration
(including in a single composition), or alternatively via different routes of
administration.
For example, the CAR T cells may be administered by injection into the blood
stream of
the subject, while the bridging molecule may be administered via another route
of
administration such as intramuscularly, intradermally, subcutaneously or
intraperitoneally.
[0432]A bridging molecule may be produced or expressed inside the body by
genetically engineered cells secreting bridging molecules spontaneously or
upon
stimulation via a stimulating agent e.g. a small molecule. Alternatively,
cells may
continuously secrete bridging molecules and will stop secreting them upon
application
of a stimulating agent, e.g. a small molecule.
[0433]
It will be clearly understood that, although this specification refers
specifically
to applications in humans, the invention is also useful for veterinary
purposes. Thus in
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all aspects the invention is useful for domestic animals such as cattle,
sheep, horses
and poultry; for companion animals such as cats and dogs; and for zoo animals.

Therefore, the general term "subject" or "subject to be / being treated" is
understood to
include all animals (such as humans, apes, dogs, cats, horses, and cows).
[0434] The term "administered" means administration of a therapeutically
effective
dose of the aforementioned composition including the respective cells to an
individual.
By "therapeutically effective amount" is meant a dose that produces the
effects for
which it is administered. The exact dose will depend on the purpose of the
treatment,
and will be ascertainable by one skilled in the art using known techniques. As
is known
in the art and described above, adjustments for systemic versus localised
delivery, age,
body weight, general health, sex, diet, time of administration, drug
interaction and the
severity of the condition may be necessary, and will be ascertainable with
routine
experimentation by those skilled in the art.
[0435] Subjects requiring treatment include those already having a benign, pre-

cancerous, or non-metastatic tumour as well as those in which the occurrence
or
recurrence of cancer is to be prevented. Subjects may have metastatic cells,
including
metastatic cells present in the ascites fluid and/or lymph node.
[0436] The objective or outcome of treatment may be to reduce the number of
cancer
cells; reduce the primary tumour size; inhibit (i.e., slow to some extent and
preferably
stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to
some extent and
preferably stop) tumour metastasis; inhibit, to some extent, tumour growth;
and/or
relieve to some extent one or more of the symptoms associated with the
disorder.
[0437] Efficacy of treatment can be measured by assessing the duration of
survival,
time to disease progression, the response rates (RR), duration of response,
and/or
quality of life.
[0438] The method is particularly useful for extending time to disease
progression.
[0439] The method is particularly useful for extending survival of the human,
including
overall survival as well as progression free survival.
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[0440] The method is particularly useful for providing a complete response to
therapy
whereby all signs of cancer in response to treatment have disappeared. This
does not
always mean the cancer has been cured.
[0441] The method is particularly useful for providing a partial response to
therapy
whereby there has been a decrease in the size of one or more tumours or
lesions, or in
the extent of cancer in the body, in response to treatment.
[0442] The objective or outcome of treatment may be any one or more of the
following:
to reduce the number of cancer cells;
reduce the primary tumour size;
inhibit (i.e., slow to some extent and preferably stop) cancer cell
infiltration into
peripheral organs;
inhibit (i.e., slow to some extent and preferably stop) tumour metastasis;
inhibit, to some extent, tumour growth;
relieve to some extent one or more of the symptoms associated with the
disorder.
[0443] In one embodiment, subjects requiring treatment include those having a
benign, pre-cancerous, non-metastatic tumour.
[0444] In one embodiment, the cancer is pre-cancerous or pre-neoplastic.
[0445] In one embodiment, the cancer is a secondary cancer or metastasis. The
secondary cancer may be located in any organ or tissue, and particularly those
organs
or tissues having relatively higher haemodynamic pressures, such as lung,
liver, kidney,
pancreas, bowel and brain. The secondary cancer may be detected in the ascites
fluid
and/or lymph nodes.
[0446] In one embodiment, the cancer may be substantially undetectable.
[0447] "Pre-cancerous" or "preneoplasia" generally refers to a condition or a
growth
that typically precedes or develops into a cancer. A "pre-cancerous" growth
may have
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cells that are characterised by abnormal cell cycle regulation, proliferation,
or
differentiation, which can be determined by markers of cell cycle.
[0448] The cancer may be a solid or a "liquid" tumour. In other words, the
cancer may
be growth in a tissue (carcinoma, sarcoma, adenomas etc) or it may be a cancer

present in bodily fluid such as in blood or bone marrow (e.g., lymphomas and
leukaemias).
[0449] In certain preferred embodiments, the cancer requiring treatment may be
a
cancer characterised by low levels of expression of dysfunctional P2X7
receptor.
Examples of such cancers include Burkitt's lymphoma. However,
immunohistochemical
analyses of surface expression of the dysfunctional P2X7 (nfP2X7) receptor on
patient
tumour biopsies reveals a range from 1+ to 3+ in IHC score. Samples with low
expression may therefore be found in a wide range of tumour types. Examples
are
found in solid tumours of various types, including but not limited to
neuroblastoma,
colorectal cancers, lung cancers, kidney cancers, skin cancers, breast
cancers, brain
cancers and prostate cancer. Such differences in expression level in different
tissues
may be due to the formation of tumours from cells that are at an earlier state
of
transformation (the tissues with the highest receptor expression may be those
undergoing the highest rate of proliferation).
[0450] Other examples of cancers that can be treated in accordance with the
methods of the present invention include blastoma (including medulloblastoma
and
retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma),
neuroendocrine tumours (including carcinoid tumours, gastrinoma, and islet
cell
cancer), nnesothelionna, schwannonna (including acoustic neuroma),
nneningionna,
adenocarcinoma, melanoma, leukaemia or lymphoid malignancies, lung cancer
including small-cell lung cancer (SCKC), non-small cell lung cancer (NSCLC),
adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the
peritoneum, hepatocellular cancer, gastric or stomach cancer including
gastrointestinal
cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer,
bladder cancer, hepatoma, breast cancer (including metastatic breast cancer),
colon
cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary
gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer,
thyroid cancer,
hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer,
oesophageal
cancer, tumours of the biliary tract, as well as head and neck cancer.
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[0451] In further examples, the methods of treatment contemplated within the
scope
of the present invention, include methods for treating or preventing an
infectious
disease. Thus, the bridging molecules of the invention can be utilised to
redirect the
CAR T cells towards an additional surface accessible antigen, for example
wherein the
antigen is a non-cancer associated pathogenic antigen presented on an MHC I or
MHC
II molecule as further described herein.
[0452] The subject requiring treatment for an infectious disease may be at
risk or
have been diagnosed with the disease. Subjects at risk include those who are
immunocompromised. Thus, the methods of the present invention also allow for
the
prevention of onset of infectious disease in individuals receiving therapy
(such as for
treating cancer) that renders them immunocompromised and therefore susceptible
to
infection.
[0453] Examples of intracellular pathogens from which peptides are presented
on
MHC I or MHC II molecules include: viral infections, intracellular bacterial
infections,
protozoan infections, and intracellular fungal infections.
[0454] Examples of viral infections that may be treated using the methods of
the
present invention include: HIV, hepatitis (e.g., Hepatitis A, B or C), a
coronavirus (e.g.
SARS-CoV-2), an influenza virus, varicella zoster virus, mumps virus.
[0455] Examples of intracellular bacterial infections which may be treated
using the
methods of the present invention include: mycobacterial infections (e.g.,
Mycobacterium
tuberculosis), Bartonella henselae, Francisella tularensis, Listeria
monocytogenes,
Salmonella Typhi, Bruce/la, Leg/one/la, Nocardia, Neisseria, Rhodococcus,
Yersinia,
Staphylococcus aureus, Chlamydia, Rickettsia, Coxiella, and Chlamydophila
pneumoniae.
[0456] Examples of intracellular infections caused by fungal pathogens:
Histoplasma
capsulatum, Cryptococcus neoformans, and Pneumocystitis jirovecii.
[0457] Examples of obligate intracellular protozoan pathogens include:
Apicomplexans (Plasmodium spp., Toxoplasma gondii and Cryptosporidium parvum),

and Trypanosomatids (Leishmania spp. and Trypanosoma cruzi).
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[0458] Immune cells that may be targeted to modulate the immune system in the
context of cancer and/or autoimmune disease may be B cells (CD19, CD20, 0D22),

plasma cells (BCMA, CD38, CD138), T cell subsets via (TRBC1 or TRBC2, a4137 &
aE137, CD7), macrophages and TAMs (0D163 and CD206). In the context of
allogeneic
stem cell transplantation, immune-based conditioning may be undertaken by
targeting
(CD34, CD117, CD133, CD33 and CD38) especially in case of non-malignant
diseases
e.g. thalassaemia major or sickle cell anaemia and/or in case of DNA-repair
defects like
Fanconi anaemia.
[0459] Targeting senescent tumour cells via the marker (uPAR) will help to
eliminate
tumour cells in a resting state and which are likely to expand at later time
points and
promote even faster proliferation of cancer cells in the latter by secreting
tumour
promoting cytokines and shaping a tumour-suppressive environment protecting
new
cancerous subclones.
[0460] CAR T cells may be constructed in a way that they are able to
immunosuppress other immune cells, e.g. TREG CAR T cells or by secreting
innnnunosuppressive cytokines (TGFbeta, IL10) and chennokines by introducing
the
corresponding inducible expression cassette [NFAT-dependent cytokine
secretion] and
the signalling in the construct.
[0461] The bridging molecules of the invention may be formulated for
administration
to a subject using techniques known to the skilled artisan. Formulations of
the bridging
molecules may include pharmaceutically acceptable excipient(s) (carriers or
diluents).
Examples of generally used excipients include, without limitation: saline,
buffered saline,
dextrose, water-for-injection, glycerol, ethanol, and conn binations thereof,
stabilising
agents, solubilising agents and surfactants, buffers and preservatives,
tonicity agents,
bulking agents, and lubricating agents.
[0462] A formulation of bridging molecules may include one type of bridging
molecule, or more than one type of bridging molecule (i.e., wherein the
bridging
molecules may have the same or different targeting and/or dysfunctional P2X7
receptor
epitope moieties).
[0463] The bridging molecules may be administered to a subject using modes and

techniques known to the skilled artisan. Exemplary modes include, but are not
limited
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to, intravenous, intraperitoneal, and intratumoural injection. Other modes
include,
without limitation, intradermal, subcutaneous (s.c, s.q., sub-Q, Hypo),
intramuscular
(i.m.), intra-arterial, intramedullary, intracardiac, intra-articular (joint),
intrasynovial (joint
fluid area), intracranial, intraspinal, and intrathecal (spinal fluids).
[0464] Formulations comprising the bridging molecule are administered to a
subject
in an amount that is effective for treating the specific indication or
disorder. In general,
formulations comprising at least about 0.01 pg/kg to about 100 mg/kg body
weight of
the bridging molecule may be administered to a subject in need of treatment.
In most
cases, the dosage may be from about 100 pg/kg to about 10 mg/kg body weight of
the
bridging molecules daily, taking into account the routes of administration,
symptoms,
etc. However, the amount of bridging molecules in formulations administered to
a
subject may vary between wide limits, depending upon the location, source,
identity,
extent and severity of the disorder, the age and condition of the individual
to be treated,
etc. A physician may ultimately determine appropriate dosages to be used. The
bridging
molecules may be administered as a continuous infusion or a bolus application.
[0465] The timing between the administration of the CAR T cell and the
bridging
molecule formulation may range widely depending on factors that include the
type of
(immune) cells being used, the binding specificity of the CAR, the identity of
the
targeting moiety and the identity of the target cell, e.g. cancer cell to be
treated, the
location of the target cell in the subject, the means used to administer the
formulations
to the subject, and the health, age and weight of the subject being treated.
Indeed, the
TCBM formulation may be administered prior to, simultaneous with, or after the

genetically engineered (immune) cell formulation.
[0466] It will be understood that the invention disclosed and defined in this
specification extends to all alternative combinations of two or more of the
individual
features mentioned or evident from the text or drawings. All of these
different
combinations constitute various alternative aspects of the invention.
Examples
Example 1 ¨ Materials and methods including generation of bridging molecules
[0467] Cultivation, transfection and protein production were performed as per
ExpiCHO Expression System User Guide (Thermo - ExpiCHOTM Expression System
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USER GUIDE. For transfection of ExpiCHO-STM Cells in a defined, serum-free
medium
Catalogue Number A29133, Publication Number MAN0014337). In summary, ExpiCHO
were routinely passaged and maintained at less than 4-6x106 cell/mL in ExpiCHO

medium. Cells in the mid-log growth phase were transfected when cell number
was in
the range of 5-7x106 cell/mL. For transfection, liposome complex was prepared
with 1
pg DNA for each mL of culture. For co-transfection of vectors coded with heavy
and
light chains separately, the vector ratio was set at 1:1 unless specified
otherwise. "High
Titer" or "Max Titer" expression protocols were followed after transfections,
and cultures
were harvested when cell viabilities dropped below 70%. Harvest was done by
centrifugation at 300xg for 5 min at 20 C. Cells were discarded and the
supernatant
was centrifuged again at 4000 x g for 30 mins at 4 C. The harvested
supernatants were
clarified by 0.2 pm filtration using PES membrane before freezing for storage.
[0468] Harvested samples could be enriched and buffer-exchanged by spin-
columns
or TFF cassette with nominal molecular size cut off of 5, 10 or 30 kDa.
[0469] For HIS-tagged column purification, the harvested supernatants were
dialysed
via SnakeSkin dialysis tube with nominal molecular size cut off 5, 10 or 30
kDa
depending on the protein of interest. For large scale production, the
supernatants were
washed through a TFF cassette with a certain molecular size cut off membrane.
Buffer-
exchange to the desired column loading buffer also was achieved through the
above-
mentioned procedures to prepare the sample for the His-tagged column
purification.
The purification was performed on either a HisTrap excel column (Cytiva) or
PureCube
100 Compact Cartridge Ni-INDOGO affinity Column (Cat#75302, Cube Biotech) or
other
equivalent column. Purification was performed on a AKTA Pure system (Cytiva)
equipped with UV detector at 280 nm wavelength, conductivity detector and pH
probe.
Loading and washing buffer consisted of 50nnM Sodium Phosphate Monobasic and
0.3
M Sodium Chloride, pH 8Ø The elution buffer contained 500mM Imidazole. The
eluted
protein was buffer exchanged to PBS using Vivaspin (Sartorius) and stored
under 4 C.
[0470] Protein was quantified via Nanodrop at 280 nm wavelength and standard
Bicinchoninic acid (BCA) protein assay. The protein purity was confirmed by
SDS PAGE
gel electrophoresis.
[0471]
The detailed experimental data generated by the inventor(s) and described
herein includes the generation of a wide variety of bridging molecules that
include:
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1. Targeting moieties generated in multiple antibody formats, e.g. Fab and
scFv;
2. Various positioning of the dysfunctional P2X7 receptor epitope moiety on
the
targeting moiety including, for example, on the VL and VH;
3. Inclusion of linkers between the targeting moiety and the dysfunctional
P2X7
receptor epitope moiety;
4. Targeting moieties that bind to a wide range of cell surface antigens that
are
present on tumour cells from different tissue origins.
Antibody/Fab conjugation
[0472] Conjugation of BILO3s 2-2-1-Fc (an anti-nfP2X7 receptor antibody) with
fluorochrome Alexa Fluor 647 (AF647) was performed according to
manufacturer's
instruction (Cat# A20186, ThermoFisher). The AF647 labelled BIL03s 2-2-1-Fc
antibody
was reconstituted in PBS, pH 7.2, with 2 mM sodium azide.
Testing binding of bridging molecules to cells by flow cytometry
[0473] Reagents
= Abs and Fabs:
= BIL03 pure antibody: prediluted to 10Oug/mL by PBS.
= BIL03-AF647 antibody: prediluted to 10Oug/mL by PBS
= Anti-His antibody-FITC (1mg/mL) (Abcam Cat# ab1206, Cat# GR3361939-1)
= Rabbit IgG-FITC isotype control (Abcam Cat# ab3706, Cat# GR3356160-1)
= Bridging molecules, generated in-house and harvested from supernatant see

below
[0474] Cell lines used for binding assays: JeKo-1 (CD19, CD20, CD79B, CD37,
CD22, ROR1, Her2), MOLM-13 (CD33, CD38, CD37, 0D135, CD123), PC3 (Her2),
MDA-MB-231 (EGFR, PD-L1), Raji (CD22, CD70, CD79B), Karpas299 (CD30), U937
(CD105), HL60, RPMI8226 (BCMA, C038, CD33).
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[0475] Cells were resuspended at a density of in 5x106 cells/ml and 100 pL
aliquots
used per well for staining (0.5x106/sample for testing).
Cell Line Disease Type Source Growth Subculture Ratio
Culture
Properties
Medium
MOLM-13 Acute Myeloid ATCC Suspension 4x105- 2x106
RPM!
Leukaemia, AML cells/mL
1640
+10%FBS
JeKo-1 Mantle Cell ATCC Suspension 2x105- 2x106
RPM!
Lymphoma, MCL cells/mL
1640
+20%FBS
MDA-MB Mammary NCI-60 Adherent 1:4
Leibovitz's
231 panel L-
15
medium +
10% FBS;
RPMI1640
+ 10%
FBS
P0-3 Prostate CellBank Adherent 1:6
F12K or
Australia
RPM!
1640 +
10% FBS
U937 Histiocytic CellBank Suspension 2x105- 9x105
RPM!
Lymphoma Australia cells/mL
1640 +
2mM
Glutamine
+ 10%
FBS
Raji Burkitt's lymphoma CellBank Suspension
3x105- 9x105 RPM!
Australia cells/mL
1640 +
2mM
Glutamine
10%FBS
HL-60 Acute NCI-60 Suspension 1x105- 1x106
IMDM
promyelocytic panel cells/mL
+10%FBS
leukaemia
RPMI- Multiple Myeloma NCI-60
Semi adhesion 5x105- 2x106 RPM!
8226 panel cells/mL
1640
+10%
FBS
Karpas299 anaplastic large CellBank Suspension 0.5-2 x106
RPM!
cell lymphoma Australia
1640 +
2mM
Glutamine
+ 10-20%
FBS
Procedure
[0476] 1. Staining condition list:
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= Bridging molecule binding to BIL03s Antibody Staining and His Tag
Antibody
detection
[0477] Control conditions:
= Control bridging molecule binding to BIL03s Antibody Staining and His Tag

Antibody detection
= No bridging molecule binding to BIL03s Antibody Staining and His Tag
Antibody
detection
= Unstained cells
[0478] 2. Staining procedure
= Block the cells with human FcR blocker (20% v/v, Miltenyi) on ice for
10min and
wash off the unbounded FCR blocker.
= Incubate the cells in 50 pL of crude prep supernatant and stain on ice
for 15min.
= Wash the cell suspension by FACS buffer x 2
= Stain the cells with 1 pg/rnL of BILO3s-AF647 and 1 pL of His Tag
antibody per
100 pL cell suspension.
= Wash the cells by FACS buffer x2.
= Cells ready to be analysed on MacsQuant16.
Lentiviral vector production using adherent Lenti-X HEK293T (Takara) cells and

PEI
Lenti-X 293T culture media
[0479] For cell culture pre transduction:
[0480] 90% Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (4.5
g/L), 4 mM L-glutamine, and sodium bicarbonate (Sigma-Aldrich, D5796); 10%
Foetal
Bovine Serum (FBS); 1 mM sodium pyruvate (Sigma-Aldrich, S8636).
[0481] For cell culture post transduction:
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[0482] 90% Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (4.5
g/L), 4 mM L-glutamine, and sodium bicarbonate (Sigma-Aldrich, D5796); 10%
Foetal
Bovine Serum (FBS); 1 mM sodium pyruvate (Sigma-Aldrich, S8636), and 10 mM
sodium butyrate.
. .õ,, õ
õõ.......................
Transfer Various
A pRSV/REV (expresses HIV-1 REV)
pMDLJRRE (expresses HIV GAG/POL)
pMD2.G (expresses VSV glycoprotein)
[0483] Protocol, Part I Lenti-X 293T cells Lentivirus Transfection
[0484] Day 0:
1. Seed 1.7 x106 cells per 15 cm dish so that they will be -
80% confluent on the
day of transfection - the following Monday (-16 x106 cells).
[0485] Day 1:
1. Check cells under microscope. The cells should be about 75-90%
confluent.
2. Gently aspirate media, add 20nnL fresh DMEM supplemented with 10% FCS
to each 15cm dish, and incubate for at least two hours before transfection.
3. Perform the transfection procedure in the afternoon (-2.30-4.30pm). Warm
an
aliquot of serum-free DMEM to 37 C.
4. Prepare the Mixture A (Plasmid DNA solution) and Mixture B (PElpro
solution).
5. Determine the required volumes of DMEM and plasnnid DNA in Mixture A
according to the table below.
147: ..-PcIP,.õ,PirPõ. T75 (75cm2) 10cm dish
6 well
Mixture A (DNA)dish (1i75cn,) (60cm2)
(9.6cm2)
Plasmid DNA* .1.:m;iP]]mi]l!MiliY.AlP 44.$.&.*IGag/pol 0
12 rig/cm'
.MM!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!E!!!!!!!!!RN!!!!!!!1
Transfer pksmid0.Ai8 g/cm2
DMEM (w/o
. 5% final
culture volume .
additives)
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6. Determine the volumes required for each plasmid DNA component for the
number of plates required. 15cm dish has an area of -175cm2.
Mixture A pg/plate # plates pg/plate
plates
CCT VGEF
Transfer plasmid 14 2
pMDL/RRE 21 7.5
pRSV/REV 10.5 7.5
pMD2.G 10.5 5
DMEM high 0.75mL 0.75mL
glucose (w/o
additives)
7. Determine the volumes required for each component of Mixture B (PElpro
solution).
For each 15cm dish,
Mixture B pL/plate # plates pL/plate #
plates
(PElpro) CCT VGEF
PElpro PElpro:DNA 56uL 22uL
1mg/mL = 1:1 ratio
DMEM high 0.75mL 0.75mL
glucose
(w/o
additives)
8. Vortex PElpro for 5 seconds and then spin down if needed to collect
liquid in
the bottom of the tube.
9. Prepare Mixture B (PElpro in media) in a 15 mL tube by adding PElpro
into the
DMEM high glucose without any additives. Add PEI into DMEM Invert up
and down a few times and spin down quickly.
10. Prepare Mixture A (plasmid DNA dilution in media) in a 50 mL conical
tube by
adding DNA into the media. Mix gently by inverting up and down and spin
down quickly.
11. Mix and prepare transfection mixture (PElpro/DNA solution) by adding
Mixture
B (PElpro solution) to Mixture A (plasmid DNA dilution). Using a p1000
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micropipette, add the PElpro solution dropwise to DNA solution and
immediately invert 3-4 times to mix. Do not vortex.
12. Incubate at room temperature for 15 min, no longer than 30min. Do not
agitate
the tube during this time.
13. After the 15-min incubation, add the transfection mixture to the
flasks/dishes
containing cells and fresh media (dropwise if possible). Mix by gently
rocking the flask/dish horizontally with back and forth and left and right
motions.
14. Incubate the flasks/dishes at 37 C with 5% CO2 overnight.
[0486] Day 2:
15. 16-18 hours after transfection, replace media. Working with 2 plates at
a
time, aspirate old media. Using a 25mL pipette, carefully add 15mL fresh
DMEM supplemented with 10% FCS, and 10mM sodium butyrate.
[0487] Day 3 first harvest 24 hour post media change:
16. Collect the supernatants from the culture flasks/dishes in 50mL Falcon
tubes.
Carefully replace each plate with 15m L fresh DMEM supplemented with
10% FCS and 10mMm sodium butyrate and return plates to incubator.
17. Centrifuge the supernatant at 500 x g for 10 min at 4C (for
concentration by
Lenti-X concentrator). For ultracentrifugation, centrifuge at 3800rpm for
30min at RT.
18. Draw up the virus-containing supernatant with a 20m L syringe and
filter
through a 0.45 pm PES filter (Millipore) into a new 50 mL Falcon tube. This
can be used as (a) Crude preparation for transduction, or proceed to (b)
concentration by ultracentrifugation, or (c) concentration by Lenti-X
concentrator. Alternatively, store crude virus 4 C overnight and pool with
48 hour harvest. Store concentrated virus in aliquots at -80 C for extended
periods.
19. Discard plates, tubes and filters in a biohazard bag in the tissue
culture hood.
Seal the biohazard bag before taking it out of the hood for disposal.
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[0488] Day 4 second harvest 48 hour post media change:
20. Collect the supernatants from the culture flasks/dishes in 50mL Falcon
tubes.
21. Centrifuge the supernatant at 2000 x g for 30 min at room temp.
22. Draw up the virus-containing supernatant with a 20mL syringe and filter
through a
0.45 pm filter (Millipore) into a new 50 mL Falcon tube. This can be pooled
with the 24hr harvest or process separately and proceed to (b)
concentration by ultracentrifugation, or (c) concentration by Lenti-X
concentrator.
23. Discard plates, tubes and filters in a biohazard bag in the tissue culture
hood.
Seal the biohazard bag before taking it out of the hood for disposal.
[0489] Part II Concentrating lentiviruses by Lenti-X concentrator
24. Harvest the lentivirus-containing supernatants. Caution: supernatants
contain live lentivirus. Pool similar stocks together, if desired. Centrifuge
briefly at 500 x
g for 10 min or filter through a 0.45 pm filter.
25. Transfer clarified supernatants to a sterile container and combine 1
volume of Lenti-X Concentrator with 3 volumes of clarified supernatant. Mix by
gentle
inversion. Larger volumes may be accommodated through the use of larger (i.e.,
250
mL or 500 mL) centrifuge tubes.
26. The incubation with the Lenti-X concentrator is done once. Either at
harvest (after 1 day) or after 2 days (pooled harvest). Incubation at least
for 30 min or
overnight and then centrifugation of the pre-incubated fluid.
27. Centrifuge samples at 1,500 x g for 45 minutes at 4 C. After
centrifugation,
an off-white pellet will be visible.
28. Carefully remove supernatant, taking care not to disturb the pellet.
Residual supernatant can be removed with either a pipette tip or by brief
centrifugation
at 1,500 x g.
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29. Gently resuspend the pellet in 1/10 to 1/100th of the original volume
using
complete DMEM, PBS, or TNE. The pellet can be somewhat sticky at first, but
will go
into suspension quickly.
30. Immediately titrate sample or store at ¨80 C in single-use aliquots.
CAR T cell generation protocol
[0490] nfP2X7 BRIDGE CAR T cells were generated by lentiviral transduction of
CD4/CD8 positive selected T cells (1:1 ratio) via magnetic activated cell
sorting (MACS)
stimulated with TransAct (all according to manufacturer's instructions)
cultivated in
1L7/1L15 supplemented TecsMACS media (both 10 ng/mL). The donor source was a
buffy coat.
[0491] CAR T cells were treated in the very same way but underwent lentiviral
transduction to express the nfP2X7 BRIDGE CAR. Activated untransduced T cells
(aUT)
do not express any receptor that can either engage with the EGFR nor the C033
bridging molecules.
[0492] Hypothesis:
[0493] nfP2X7 BRIDGE CAR T cells have a superior effector function over aUT as

they are redirected towards cancer cells directly via nfP2X7 recognition on
the cell
surface of MOLM-13 leukaemic cells.
[0494] nfP2X7 BRIDGE CAR T cells have a superior effector function over aUT as

they are redirected towards cancer cells indirectly via nfP2X7 E200 derived
epitope on
the 0D33 Fab-bridging molecules on the surface of MOLM-13 leukaemic cells.
Reagent and equipment preparation
[0495] CAR T culture medium: TexMACS with human IL-7 and IL-15. IL-7 stock
concentration was 100 ug/mL, each vial has 55 pL. IL-15 stock concentration is
50
pg/mL, each vial had 55 pL.
[0496] For preparation of TexMACS with final concentration of 10 ng/mL of IL-
7,
5ng/mL of IL-15 and 3% FBS, add 50u1 of IL-7, 50 ul of IL-15 stock, and 15 mL
FBS into
each bottle (500 mL) of TexMACS medium. Label the date of adding of cytokines
on the
medium bottle.
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[0497] Freezing medium preparation on the day of harvest: 10% of DMSO, 90% of
FBS. Note: Add the reagent into 50mL falcon tube according to the following
order:
DMSO to FBS.
Part l: T cell activation and T cell transduction
[0498] Day 1: T cell activation
1. CD4+ and CD8+ CAR T cells separation from whole blood or buffy coat.
Refer to protocol of PBMC separation and CD4 and CD8 cell separation.
2. Wash the CD4+ and CD8+ cells twice with pre-warmed TexMACS
Medium (without supplement cytokines) by filling the falcon tube to the
maximum
volume and centrifuge at 300x g for 5 minutes. Aspirate supernatant
completely.
3. Resuspend the CD4+ and CD8+ cells in pre-warmed TexMACS medium
supplemented with 10 ng/mL IL-7, 5 ng/mL IL-15 and 3% FBS to a final
concentration of
10^6 cells/mL.
4. Plate the CD4+ cells and the CD8+ cells with a ratio of 1:1 by adding
0.5 ml of CD4+ cells and 0.5 mL of CD8+ cells into each well in a 24 well
plate.
5. Add 10 pL of T cell TransAct to a final dilution of 1:100 in the cell
culture
and carefully resuspend.
6. Incubate for ¨36 hours at 37 C with 5% CO2 before transduction.
[0499] Day 3: T cell Transduction
7. Use fresh viral vectors for transduction if possible. Otherwise, thaw
slowly
frozen viral vectors on ice.
8. Remove 800 pL of the media slowly using a P1000 pipette from the side of

the wells, taking care not to disrupt the bottom cell layer.
9. Add in dropwise 150 pL (one plate's worth of viral vectors) or 300 pL (2
plates' worth of viral vectors) to the T cells. Top up the wells with fresh
supplemented
TexMACS media up to 600 pL and add polybrene to each well at a final
concentration
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of 4 pg/mL. From this time onwards, the T cells should be kept in an incubator
for
lentiviral work only.
10. After transduction, appropriate clean up procedures should be performed.
Decontaminate the biosafety cabinet and the aspirator line by cleaning
surfaces and
running the line with 2% Virkon solution following by 70% ethanol. Dispose of
contaminated waste, such as tips and serological pipettes etc. in a biohazard
bag inside
of the biosafety cabinet, seal the bag before taking the waste outside for
disposal.
[0500] Day 4 onwards ¨ T cell maintenance
11. Transduced T cells were maintained at in IL-7, IL-15 and 3% FBS
containing TexMACS media.
12. Observe the T cell growth in the 24 well plate, transfer the cells to T75
flasks on day 2 post transduction.
13. Monitor the lactate levels in the media using CCS Analyzer daily.
Replenish fresh media if the lactate level is >10mmol/L; Ideally, keep the
lactate level
<4m mol/L.
[0501] Day 9 ¨ Day 5 post T cell transduction ¨ Expression analysis
[0502] Transduced T cells are counted on day 5 using a flow cytometer. A
sample is
taken for flow cytometry analysis to determine expression efficiency based on
a
standard flow cytometry protocol.
Luciferase expressing cancer cell line generation
[0503] Firefly luciferase lentiviral transfer plasmids used for
viral vector production:
= pRRLsin18. cPPT.EF1a_firefly_l uciferase_T2A_EGFP.WPRE
- pRRLsin18. cPPT.hPGK_firefly_luciferase_T2A_EGFP.WPRE
1. Plate cells at a density of 300,000 cells/well in a 24-well plate in a
total
volume of 850 pL.
2. Viral vectors containing the firefly luciferase gene are produced using a 4-

plasmid transfection protocol as described in the lentiviral production
section. Use fresh
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concentrated viral vectors for transduction if possible. Otherwise, thaw
slowly frozen
viral vectors on ice.
3. Add in dropwise 150 pL (one plate's worth of viral vectors) to the
cells.
From this time onwards, the cell should be kept in an incubator for lentiviral
work only.
4. Incubator cells at 37 C with 5% CO2.
5. After transduction, appropriate clean up procedures should be performed.
Decontaminate the biosafety cabinet and the aspirator line by cleaning
surfaces and
running the line with 2% Virkon solution following by 70% ethanol. Dispose of
contaminated waste, such as tips and serological pipettes etc. in a biohazard
bag inside
of the biosafety cabinet, seal the bag before taking the waste outside for
disposal.
6. On day 2 post transduction, transfer cells to 125 flasks.
7. On day 5 post transduction, count cells and take a sample for expression

analysis by flow cytometry.
8. On day 7 post transduction, transduced cells are bulk sorted based on
eGFP expression using a live cell sorter.
9. After expansion, bulk sorted cells are further sorted by single cell
clones.
Single cell clones are grown, expanded and frozen down to make stocks.
Functional assays
[0504] Target cells constitutively expressing firefly luciferase and eGFP
(sorted and
grown as single cell clones defined as high performance cell lines) were used
in the
functional assays to measure viability via bioluminescence and/or
fluorescence. The
amount of light emitted correlates to the total number of cells in
bioluminescence and
the fluorescent target cells identified via flow cytometry correlate with the
total number
of cells alive.
[0505] Effector and target cells were seeded according to indicated effector
to target
ratio (ET). The indicated ET ratio, e.g. 10:1 is always referred to the total
number of T
cells and the total number of target cells. As the CAR expressing fraction is
different
from the total number of T cells the ET ratio referred to the CAR expressing
cells is
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indicated separately. Target cells were seeded with 25,000 or 50,000 cells per
96 well
plate.
Luciferase killing assay
[0506] Effector and target cells were seeded according to indicated effector
to target
ratios (ET). The BRIDGE molecules were added in the indicated format (Fab,
IgG1) at
the indicated concentrations. D-luciferin was added and bioluminescence was
measured at the indicated time points after incubation was started under
standard
conditions in incubators at 37 C and 5% CO2 on a SpectraMaxi3.
[0507] Viability of cells was calculated according to a serial dilution
derived
bioluminescence activity curve of cells (100%, 75%, 50%, 25%, 10% and 0%
target
cells) and depicted in percent viable cells. In general, the lysis was
calculated by
(bioluminescence of testing condition ¨ 0% bioluminescence) / (100%
bioluminescence
¨ 0% bioluminescence).
Flow based kill assay
[0508] Effector and target cells were seeded according to indicated effector
to target
ratios (ET), the BRIDGE molecules were added in the indicated format (Fab,
IgG1) at
the indicated concentrations. Cell number was measured at the indicated time
points
(24h or 48h) after incubation was started under standard conditions in
incubators at
37 C and 5% CO2 on a MACSQuant16 flow cytometer according to standard
protocols.
The staining of cells included a viability dye to exclude all dead cells from
the analysis.
T cells were clearly differentiated from eGFP positive cancer cells via CD3.
Further T
cells were characterised by CD25 and CD69 as a measure for specific T cell
activation
according to standard protocols after 24h or 48h. The final data analysis was
performed
by FlowJo10.
[0509] Antibody cocktail
Channel Antibody Source Cat#
R1 CD3 APC Miltenyi 130-113-135
R3 CD25 APCVio770 Miltenyi 130-123-469
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V2 CD69 VioGreen Miltenyi 130-112-611
B1 eGFP Constitutive stable Target cell
marker
expression
V1 Viobil ity405/452 Miltenyi 130-109-816
Flow based acquisition of cytokine secretion
[0510] Effector and target cells were seeded according to indicated effector
to target
ratios (ET), the BRIDGE molecules were added in the indicated format (Fab,
IgG1) at
the indicated concentrations. Supernatant was collected after 24h or 48h and
measured
at the indicated time points after incubation was started under standard
conditions in
incubators at 37 C and 5% CO2 on a MACSQuant16 flow cytometer according to
standard protocols using the Miltenyi cytokine beads. The final data analysis
was
performed by FlowJo10.
Example 2 ¨ Characterisation of various bridging molecules
[0511] The results in Figures 4 to 7 show that bridging molecules comprising a

targeting moiety in the form of a Fab or scFv (FMC63 clone; amino acid
sequences
described herein) that can bind to CD19, binds to CD19 on the surface of live
cells and
can present the dysfunctional P2X7 receptor epitope moiety (e.g. E200 moiety)
such
that it is accessible by an anti-P2X7 receptor antibody (BIL03s 2-2-1-Fc). The
location of
the dysfunctional P2X7 receptor epitope moiety can vary and the targeting
moiety can
still bind to its target cell surface antigen and the dysfunctional P2X7
receptor epitope
moiety is still available for binding to an antibody. Note that BILO3s 2-2-1-
Fe ¨ AF647 /
HIS - FITC did not bind to control bridging molecules that did not contain the

dysfunctional P2X7 receptor epitope moiety, nor did the anti-HIS antibody bind
to control
bridging molecules that did contain a HIS tag (data not shown).
Fab format +/-linker¨ epitope on VH
[0512] Figure 4 shows that bridging molecules in Fab format with a single E200

epitope either directly linked to the VH or via a linker binds to CD19 on JeKo-
1 (mantle
cell lymphoma) cell line and the E200 epitope is available for binding to an
antibody.
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scFV format +/-linker¨ epitope on VH
[0513] Figure 5 shows that bridging molecules in scFv format with a single
E200
epitope either directly linked to the VH or via a linker binds to CD19 on JeKo-
1 (mantle
cell lymphoma) cell line and the E200 epitope is available for binding to an
antibody.
Fab format +/-linker¨ epitope on VL
[0514] Figure 6 shows that bridging molecules in Fab format with a single E200

epitope either directly linked to the VL or via a linker binds to CD19 on JeKo-
1 (mantle
cell lymphoma) cell line and the E200 epitope is available for binding to an
antibody.
scFv format +/-linker¨ epitope on VL
[0515] Figure 7 shows that bridging molecules in scFv format with a single
E200
epitope either directly linked to the VL or via a linker binds to CD19 on JeKo-
1 (mantle
cell lymphoma) cell line and the E200 epitope is available for binding to an
antibody.
Bridging molecules targeting various antigens
[0516] Figure 8: Binding of bridging molecules to various antigens CD37,
CD79B,
ROR1, 0D33, CD38, 0D123, 0D135, BCMA, EGFR, PDL1, CD22, CD70 and CD20.
(a), (c), (e), (g), (i), (k), (m), (o), (q), (s), (u), (w) and (y) show anti-
HIS antibody binding,
(b), (d), (f), (h), (j), (I), (n), (p), (r), (t), (v), (x) and (z) show
binding of antibody to
dysfunctional P2X7 receptor epitope.
[0517] CD37 targeting moieties derived from otlertuzumab, CD79B targeting
moieties
derived from polatuzumab, ROR1 targeting moieties derived from ROR1 APC
(W02016016344A1_D10v3), CD33 targeting moieties derived from lintuzumab, 0038
targeting moieties derived from daratumumab, C0123 targeting moieties derived
from
clone 32716, 0D135 targeting moieties derived from 4G8, BCMA targeting
moieties
derived from clone CA8 J9M0, EGFR targeting moieties derived from necitumumab
or
matuzumab, PDL1 targeting moieties derived from atezolizumab, CD22 targeting
moieties derived from m971-L7 (or inotuzumab (data not shown)), CD70 targeting

moieties derived from cusatuzumab, CD19 targeting moieties derived from
tafasitamab,
CD20 targeting moieties derived from ofatumumab (or ocrelizumab (data not
shown)).
Exemplary amino acid sequences of bridging molecules targeting these antigens
and
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derived from the antibodies mentioned immediately above are described in the
sequence information table herein.
[0518]
Figure 8 shows binding of various bridging molecules to JeKo-1 (MCL) wild
type cell line (CD37, CD79B, ROR1) Rail (Burkitt's lymphoma) wild type cell
line (CD22,
CD70, CD19, CD20, CD22), MOLM-13 (AML) wild type cell line (CD33, CD38, CD123
and CD135), RPM! 8226 (multiple myeloma) wild type cell line (CD33, BCMA and
CD38), MDA-MB 231 (breast cancer) wildtype cell line (EGFR and PDL1) and PC-3
(prostate cancer) wild type cell line (EGFR).
Example 3 ¨ Dose dependent increasing in cell binding by CD19 targeting
bridging molecule
[0519] JeKo-1 (MCL) CRL3006TM wild type cell line purchased from ATCC as part
of
the NCI60 panel. The cells were cultured according to general recommendations
and
standards for this particular cell line.
[0520] Figure 9 shows "painting" of JeKo-1 with CD19 targeted Fab bridging
molecules in the illustrated format.
[0521] Cells were incubated at indicated concentrations with Fab bridging
molecules.
CD33-targeted Fab-bridging molecules served as negative control in JeKo-1 at
10
ng/mL and 1000 ng/mL. CD19 targeted Fab-bridging molecule were used at 1
ng/mL,
ng/mL, 100 ng/mL and 1000 ng/mL.
[0522] The flow cytometric staining was undertaken in two steps according to
standards in flow cytometric staining using the Fc block reagent (Miltenyi).
First the
target cells were incubated with Fab-bridging molecules at indicated
concentrations for
min, washed three times and then the secondary antibodies anti-HIS FITC and
the
single domain antibody BIL03 2-2-1 AF647 was used at saturating concentrations
(1
ug/mL) to indirectly stain the target cells via the 6xHIS and the nfP2X7 E200
derived
epitope on the bound Fab-bridging molecules. After 15 min of incubation the
sample
was washed and then analysed on a MACSQuant16 (Miltenyi). The flow data was
analysed via FlowJo v10.7 (BD).
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[0523] There is no expression of CD33 in JeKo-1 cells. CD19 staining showed
increasing expression with increasing concentrations of CD19-targeted bridging

molecules.
Example 4 ¨ Dose dependent increase in cell bindina by CD33 taraetina bridaina

molecule
[0524] MOLM-13 (AML) wild type cell line purchased from ATCC as part of the
NCI60
panel. The cells were cultured according to general recommendations and
standards for
this particular cell line.
[0525] Figure 10 "painting" of MOLM-13 with CD33 targeted Fab bridging
molecules
in the illustrated format.
[0526] Cells were incubated at indicated concentrations with Fab bridging
molecules.
CD19 targeted Fab bridging molecule served as negative control in MOLM-13 at
10
ng/mL and 1000 ng/mL, while CD33 targeted Fab-bridging molecule was used at 1
ng/mL, 10 ng/mL, 100 ng/mL and 1000 ng/mL.
[0527] The flow cytometric staining was undertaken in two steps according to
standards in flow cytometric staining using the Fc block reagent (Miltenyi).
First the
target cells were incubated with Fab-bridging molecules at indicated
concentrations for
15 min, washed three times and then the secondary antibodies anti-HIS FITC and
the
single domain antibody BIL03 2-2-1 AF647 was used at saturating concentrations
(1
ug/mL) to indirectly stain the target cells via the 6xHIS and the nfP2X7 E200
derived
epitope on the bound Fab-bridging molecules. After 15 min of incubation the
sample
was washed and then analysed on a MACSQuand16 (Miltenyi). The flow data was
analysed via FlowJo v10.7 (BD).
[0528] There is no expression of CD19 in MOLM-13 cells. CD33 staining showed
increasing expression with increasing concentrations of CD33-targeted bridging

molecules.
Example 5 - Detection of marker genes and direct CAR detection via nfP2X7 E200

derived peptide and bridging molecule
[0529] nfP2X7 targeted bridging CAR effector cells (such as T cells or NK
cells but not
limited to) recognise cancer cells specifically. The epitope targeted in
nfP2X7 arises
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from a conformational change of the P2X7 protein trimer and is exposed solely
on
cancer cells and not exposed on healthy cells.
[0530] The basic principle as well as the engagement of nfPX7 CAR expressing
effector cells via nfP2X7 E200 derived peptide tagged bridging molecules and
the
different formats of bridging molecules is illustrated and outlined in Figures
1 to 3.
[0531] Thus using nfP2X7 CAR in the absence of bridging molecules, the nfP2X7
CAR expressing effector cells exhibit cancer-specific targeting.
[0532] In order to broaden the applicability to nfP2X7 functionally negative
cancers
(very low or possibly negative for nfP2X7) nfP2X7 CAR expressing effector
cells may be
redirected to cancer cells via bridging molecules targeting cancer-associated
antigens
as illustrated for CD33 or cancer-specific antigens via TcR-like mAbs. The
specificity of
the bridging molecules is unlimited, which means any surface expressed target
antigen
or presented antigen in the context of MHC peptide presentation (class I and
II) via TcR-
like mAb or ligands may engage the nfP2X7 bridging CAR expressing effector
cells in
the same mode of action.
[0533] In most cases, the dual-function of the nfP2X7 bridging CAR expressing
effector cells is utilised. It is a combination of scenario I and ll which
means that nfP2X7
bridging CAR expressing effector cells are engaged directly to cancer cells
via nfP2X7
expressed on the cancer cells and additionally recruited to the cancer cells
via bridging
molecules targeting cancer-associated antigens as illustrated for CD33 or
cancer-
specific antigens via TcR-like mAbs.
Example 6 - "Painting" of MOLM-13 (AML) wildtvpe cell line with CD33 targeted

Fab-bridging molecules
[0534] Figure 11 illustrates the "painting" of MOLM-13 (AML) cells via CD33
targeted
Fab-bridging molecules. The flow data shows isotype control (black), staining
with
BILO3s 2-2-1-Fc sd-nnAb only at 1 ug/mL (blue) and the increase of staining
via the
combination of C033 targeted Fab-bridging molecules and BILO3s 2-2-1-Fc
(green).
The increase of target molecules that can be recognised by the nfP2X7 bridging
CAR
expressing effector cells translates into CAR-mediated effector function. The
bridging
technology therefore enhances the CAR function by increasing the targeting
epitopes
on the cancer cells.
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Example 7 - Titration of CD33 targeted bridging molecule derived from
Lintuzumab
[0535] MOLM-13 cells constitutively expressing Luciferase were co-incubated
with
increasing concentrations of CD33 targeted Fab-bridging molecules at the
indicated
concentrations. The Fab-bridging molecules bound to CD33 on MOLM-13 but do not

exert any direct toxicity arising from complement-dependent cytotoxicity (CDC)
or
recruitment of effector cells (ADCC). No cells other than MOLM-13 were used in
the
assay. The CD33 targeted Fab-bridging molecules do not have any functional
sites for
CDC or ADCC like full-size antibodies.
[0536] There was no significant impact on viability at 4 hours and no
consistent or
dose-dependent toxicity after 24 hour incubation.
Example 8¨ Activation of nfP2X7CAR-T cells by CD33-targeting Fab-bridging
molecules
[0537] Figure 12 shows a representative flow cytometric plot with direct
comparison
of untransduced T cells (left panel), CAR0007_hPGK (middle panel; CAR0007 is
also
referred to herein as CAR7) and CAR0007_EF1a (right panel; CAR0007 is also
referred
to herein as CAR7) expressing T cells. Both CAR T cells generated increased
expression of the activation markers CD25 and 0069 in incubation with MOLM-13
at
20:1 ET ratio and CD33 targeted Fab-bridging molecules at 1000 ng/mL after 48
hours.
[0538] The 0D33 Fab bridging molecule used in the experiments in Examples 8
and
9 was derived from Lintuzumab and the EGFR targeted bridging molecule derived
from
Necitumumab. The dysfunctional P2X7 receptor epitope was linked directly to
the VL
with a free N-terminus.
[0539] The CAR0007_hPGK CAR has the following domain structure from N-
terminus to C-terminus: hPGK ¨ CD8a SP - VH BIL03 2-2-1 - 0028 - CD28T - 0028 -

CD137 - CD3zeta - T2A ¨ tEGFR.
[0540] CAR0007_EF1a CAR has the following domain structure from N-terminus to
C-terminus: EF1a ¨ CD8a SP - VH BIL03 2-2-1 - 0D28 - CD28T - 0D28 - CD137 -
CD3zeta - T2A ¨ tEGFR.
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Example 9¨ Clearance of leukaemic cells by nfP2X7 CAR-T cells in the presence
of C033 bridoino molecules
[0541] The flow cytometric plots of T cells and MOLM-13 at 20:1 ET ratio and
CD33
targeted Fab-bridging molecules at 1000 ng/mL after 48 hours (corresponding to
Figure
12) showed a complete clearance of leukaemic cells in the presence of
CAR0007_hPGK and CAR0007_EF1a whereas there was no impact on leukaemic cell
number in the untransduced T cells (Figure 13).
[0542]
Figure 14 shows flow cytometric plots that illustrate the dose-dependent
clearance of leukaemic cells at indicated concentrations of 0033 targeted Fab
bridging
molecule concentrations of CAR0007_EF1a containing T cells and MOLM-13 at 20:1

ET ratio after 48 hours. Concentrations as low as 40 ng/mL showed almost
complete
elimination of leukaemic cells and complete elimination of leukaemic cells at
200 and
1000 ng/mL.
[0543] Specific lysis of MOLM-13 leukaemic cells by CAR0007_hPGK T cells at an

ET ratio of 20:1 after 48 hour incubation with and without EGFR and 0033
targeted
bridging molecules at indicated concentrations is illustrated in Figure 15(a).
Significant
lysis in a dose dependent manner was found for increasing concentrations (40,
200 and
1000 ng/mL) of C033 targeted Fab bridging molecules.
[0544] Specific lysis of MOLM-13 leukaemic cells by CAR0007_hEF1a T cells at
an
ET ratio of 20:1 after 48 hour incubation with and without EGFR and C033
targeted
bridging molecules at indicated concentrations is illustrated in Figure 15(b).
Significant
lysis in a dose-dependent manner was found for increasing concentrations (40,
200 and
1000 ng/mL) of 0033 targeted Fab bridging molecules.
[0545] In a titration experiment EGFR and CD33 targeted Fab-bridging molecules

were used to test the impact on killing of MOLM-13 by CAR0007_hPGK (Figure
16).
There was a significant difference in the viability of MOLM-13 after 24 hour
incubation at
10:1 ET ratio between EGFR targeted bridging molecules and C033 targeted
bridging
molecules at 1000 ng/mL. Statistical analysis was done by t-test. Alternative
representation of the data from Figure 16, shown in Figure 17. There was no
significant
difference in the titration of the EGFR bridging molecules (data not shown).
There were
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significant differences in the titration of the CD33 bridging molecules.
Statistical
analyses were performed by One-Way ANOVA and post-hoc test Tukey.
[0546] In a titration experiment, EGFR and C033 targeted Fab-bridging
molecules
were used to test the effect of CAR0007_hPGK on killing of MOLM-13 (Figure
18).
There was a significant difference in the viability of MOLM-13 after 24 hour
incubation at
20:1 ET ratio between the condition with EGFR targeted bridging molecules and
C033
targeted bridging molecules at 200 ng/mL and 1000 ng/mL. Statistical analysis
was
done by t-test. Alternative representation of the data from Figure 18, shown
in Figure
19. There was no significant difference in the titration of the EGFR bridging
molecules
(data not shown). There were significant differences in the titration of the
CD33 bridging
molecules. Statistical analyses were performed by One-Way ANOVA and post-hoc
test
Tukey.
[0547] In a titration experiment, EGFR and CD33 targeted Fab-bridging
molecules
were used to test the impact on killing of MOLM-13 by CAR0007_hPGK (Figure
20).
There was a significant difference in the viability of MOLM-13 after 48 hour
incubation at
10:1 ET ratio between EGFR targeted bridging molecules and CD33 targeted
bridging
molecules at 200 ng/mL and 1000 ng/mL. Statistical analysis was performed by t-
test.
[0548] In a titration experiment, EGFR and CD33 targeted Fab-bridging
molecules
were used to test the difference in killing of MOLM-13 by CAR0007_hPGK (Figure
21).
There was a significant impact on the viability of MOLM-13 after 48 hour
incubation at
20:1 ET ratio between EGFR targeted bridging molecules and CD33 targeted
bridging
molecules at 200 ng/mL and 1000 ng/mL. Statistical analysis was performed by t-
test.
[0549] In a titration experiment, EGFR and CD33 targeted Fab-bridging
molecules
were used to test the effect of CAR0007_EF1a on killing of MOLM-13 by (Figure
22).
There was a significant difference in the viability of MOLM-13 after 24 hour
incubation at
10:1 ET ratio between EGFR targeted bridging molecules and 0D33 targeted
bridging
molecules at 200 ng/mL and 1000 ng/mL. Statistical analysis was done by t-
test.
Alternative representation of the data from Figure 22, shown in Figure 23.
There was no
significant difference in the titration of the EGFR bridging molecules (data
not shown).
There were significant differences in the titration of the CD33 bridging
molecules.
Statistical analyses were performed by One-Way ANOVA and post-hoc test Tukey.
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[0550] In a titration experiment, EGFR and CD33 targeted Fab-bridging
molecules
were used to test the effect of CAR0007_EF1a on killing of MOLM-13 by (Figure
24).
There was a significant difference in the viability of MOLM-13 after 24 hour
incubation at
20:1 ET ratio between the EGFR targeted bridging molecules and CD33 targeted
bridging molecules at 40 ng/mL, 200 ng/mL and 1000 ng/mL. Statistical analysis
was
done by t-test. An alternative representation of the data from Figure 24 is
shown in
Figure 25. There was no significant difference in the titration of the EGFR
bridging
molecules (data not shown). There were significant differences in the
titration of the
CD33 bridging molecules. Statistical analyses were performed by One-Way ANOVA
and post-hoc test Tukey.
[0551] In a titration experiment EGFR and C033 targeted Fab-bridging molecules

were used to test the impact on killing of MOLM-13 by CAR0007_EF1a (Figure
26).
There was a significant impact on the viability of MOLM-13 after 48 hour
incubation at
10:1 ET ratio between the condition with EGFR targeted bridging molecules and
C033
targeted bridging molecules at 40 ng/mL, 200 ng/mL and 1000 ng/mL. Statistical

analysis was performed by t-test.
[0552] In a titration experiment, EGFR and 0033 targeted Fab-bridging
molecules
were used to test the effect of CAR0007_EF1a on killing of MOLM-13 by (Figure
27).
There was a significant difference in the viability of MOLM-13 after 48 hour
incubation at
20:1 ET ratio between the EGFR targeted bridging molecules and CD33 targeted
bridging molecules at 40 ng/mL, 200 ng/mL and 1000 ng/mL. Statistical analysis
was
performed by t-test.
[0553] In a titration experiment, EGFR targeted Fab-bridging molecules were
used to
test the effect of untransduced T cells, CAR0007_hPGK and CAR0007_EF1a
effector
cells on the killing of MOLM-13 at an ET ratio 10:1 after 24 hour incubation.
There was
no significant difference in the titration of the EGFR bridging molecules
between the
three effector cell populations (data not shown).
Example 10¨ Activation of various nfP2X7 CAR-T cells by CD33-targeting Fab-
bridging molecules - titration
[0554] In a flow based assay, activation was assessed by measuring the up-
regulation of 0025 and 0069 expression on MOLM-13 induced by untransduced T
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cells, and CAR0007_hPGK and CAR0007_EF1a expressing T cells at a 20:1 ET ratio

after 48 hour incubation both with and without EGFR or CD33 targeted Fab-
bridging
molecules.
[0555] Both the CAR0007_hPGK as well as CAR0007_EF1a expressing T cells
showed a significantly increased expression of the activation markers CD25 and
CD69
in the presence of EGFR and the C033 bridging molecules compared to
untransduced
T cells (data not shown).
Example 11 ¨ Cell killing by various nfP2X7 CAR-T cells in the presence of
CD33-
targeting Fab-bridging molecules -titration
[0556] In a cell killing assay, cytotoxicity was measured by quantification of
residual
leukaemic cells compared with a control. Elimination of leukaemic cells at
concentrations of 40, 200 and 1000 ng/mL of the EGFR bridging molecules showed

there was no significant difference between untransduced T cells compared with
CAR T
cells (Figure 28(a)). However, the elimination of leukaemic cells in the
presence of
CD33 bridging molecules at the same concentrations of 40, 200 and 1000 ng/mL
showed a significant difference between untransduced T cells compared with
both
CAR0007 transduced T cells (hPGK and EF1a) (Figure 28(b)).
Example 12 ¨ Cell killing by various nfP2X7 CAR-T cells in the presence of
C033-
targeting Fab-bridging molecules -titration
[0557] In a titration experiment, C033 targeted Fab-bridging molecules were
used to
test the effect of untransduced T cells, CAR0007_hPGK and CAR0007_EF1a
effector
cells on the killing of MOLM-13 by at an ET ratio of 10:1 after 48 hour
incubation (Figure
29). There was a consistent significant difference observed between the three
effector
cell populations resulting from titrating the CD33 bridging molecules over the

concentration range 40 ng/mL, 200 ng/mL and 1000 ng/mL (Figure 30).
Statistical
analysis was done by One-Way ANOVA and post-hoc test Tukey to compare the
effects
of the named three effector cell types compared with the concentration of EGFR

bridging molecules.
[0558] In a titration experiment, CD33 targeted Fab-bridging molecules were
used to
test the effect of untransduced T cells, CAR0007_hPGK and CAR0007_EF1a
effector
cells on the killing of MOLM-13 at an ET ratio of 20:1 after 48 hour
incubation (Figure
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31). There was a consistent significant difference observed between the three
effector
cell populations resulting from titrating the CD33 bridging molecules over the

concentration range 40 ng/mL, 200 ng/mL and 1000 ng/mL (Figure 32).
Statistical
analyses were performed by One-Way ANOVA and post-hoc test Tukey to compare
the
effects of the named three effector cell types compared with the concentration
of EGFR
bridging molecules.
Example 13 - Cell killing by nfP2X7 CAR-T cells in the presence of CD19-
targeting
bridging molecules in Fab and IgG1 format and with different dysfunctional
P2X7
receptor epitope moieties
[0559] Bridging molecules were generated with CD19 binding targeting moieties
in
different formats ¨ Fab and IgG1. Moreover, those bridging molecules in those
different
formats were generated with different dysfunctional P2X7 receptor epitope
moieties.
[0560] In these experiments the nfP2X7 CAR was as per CAR0007_EF1a CAR but
with a CD8alpha spacer between the VH BIL03 2-2-1 and the CD28TM (described
herein as CAR10).
[0561] These CD19 targeted Fab or IgG1-bridging molecules were used to test
the
impact on killing of JeKo-1 by CAR10 (Figure 33; Fab formats shown in (a),
IgG1
formats shown in (b)). There was a significant difference in the viability of
JeKo1 after,
at least, a 24 hour incubation at 10:1 ET ratio between all CD19 targeted
bridging
molecules at 100 ng/mL that contain a dysfunctional P2X7 receptor epitope
moiety (ie
0R19_8, 10 and 11) compared with the control bridging molecule that does not
have a
dysfunctional P2X7 receptor epitope moiety (OR19_7). Statistical analysis was
performed by One-Way ANOVA and post-hoc test Tukey. There was no significant
difference in the titration of the CD19 bridging molecules containing a
dysfunctional
P2X7 receptor epitope moiety. Similar results were achieved with other nfP2X7
CARs
(data not shown).
[0562] These data show that the cell killing of CAR-T cells can be potentiated

significantly by bridging molecules irrespective of targeting moiety format
and
dysfunctional P2X7 receptor epitope moiety.
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Example 14 - Cell killing by nfP2X7 CAR-T cells in the presence of CD19-
targeting
bridging molecules in Fab format and with different dysfunctional P2X7
receptor
epitope moieties
[0563] Similar experiments were performed as described above in Example 13,
however CD19 bridging molecules with a larger array of dysfunctional P2X7
receptor
epitope moieties were tested. As shown in Figure 34, CD19 bridging molecules
comprising a dysfunctional P2X7 receptor epitope moiety significantly reduced
the cell
viability of the target JeKo-1 cells whereas the control bridging molecule
without a
dysfunctional P2X7 receptor epitope moiety (0R19_7), did not.
[0564] A summary of the various Fab bridging molecules with
different dysfunctional
P2X7 receptor epitope moieties (or the absence of) is shown below (all Fab
light chains
are paired with the Fab heavy chain described herein - CD19, tafasitamab, B020-
2_HC,
SEQ ID NO: 52/143)
[0565] Table 3:
Bridging Fab Light chain
dysfunctional Moiety SEQ ID NO
molecule SEQ ID NO P2X7 receptor
epitope
moiety
0R19_7 144 None -
OR19_8 144 E200 (C to S) 4
0R19_9 144 E200_G4S 15
0R19_10 144 E200 3*G4S 168
0R19_11 144 E200_extended 10
peptide 17(27
aa)
0R19_12 144 E200_extended 6
peptide 17v2
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(22 aa)
0R19_13 144 E200_extended 17
peptide 17v3
(24 aa)
0R19_14 144 E200_extended 18
peptide 17v4
(22 aa)
0R19_15 144 E200_extended 19
peptide 17v5
(19 aa)
0R19_NEVV_001 144 E200_extended 20
peptide 17v6
(22 aa)
0R19_NEVV_002 144 E200_extended 21
peptide 17v7
(25 aa)
0R19_NEVV_003 144 E200_extended 22
peptide 17v8
(30 aa)
0R19_NEVV_004 144 E200_extended 23
peptide 17v9
(35 aa)
0R19_NEVV_005 144 E200_extended 24
peptide 17v10
(30 aa)
0R19_NEVV_006 144 E200_extended 25
peptide 17v11
(32 aa)
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0R19_NEW_007 144 E200 extended 26
peptide 17v12
(35 aa)
0R19_NEW_008 144 E200 extended 27
peptide 17v13
(25 aa)
0R19_NEW_009 144 E200 extended 28
peptide 17v14
(22 aa)
0R19_NEW_010 144 E200 extended 29
peptide 17v15
(30 aa)
0R19_NEW_011 144 E200 extended 30
peptide 17v16
(27 aa)
0R19_NEW_012 144 E200_2*G4S 16
Example 15¨ Cell killing by different nfP2X7 CAR-T cells in the presence of
CD19-
targeting bridging molecules in Fab or IgG format
[0566] Similar experiments were performed as described above in Example 13,
using
CAR-T cells having one of three different CAR architectures. As shown in
Figure 35,
three different CARs (CAR7, CAR10 and CAR16), when expressed on T cells, and
in
combination with CD19 bridging molecules comprising a dysfunctional P2X7
receptor
epitope moiety, significantly reduced the cell viability of the target JeKo-1
cells. In all
experiments, the bridging molecule 0R19_10 was used.
[0567] These results demonstrate that the bridging molecule system is
effective at
reducing cell viability when using CAR-T cells having differing CAR
architectures.
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Example 16 ¨ Binding and cell killing using alternative bridging molecule
constructs
[0568] The inventors developed a further generation of bridging molecules,
this time
comprising IgG1 hinge regions in the region linking the E200 epitope to the
targeting
moiety. The sequences of the tumour-specific antigen epitope moiety of these
bridging
molecules is provided in SEQ ID NOs: 365 to 400 (correlating to epitope
binding moiety
of the bridges referred to as 61 to B36 in Table 4 below). The tumour-specific
antigen
epitope moieties were linked to anti-CD19 or anti-0033 Fabs, as described
herein in
Table 1.
[0569] The bridging molecules were then assessed for their ability to bind
both anti-
nfP2X7 CARs and target cells (ie cells expressing the antigen to which the
targeting
moiety is designed). As summarised in the table below, all anti-CD19 bridging
molecules were determined to be able to specifically bind to Jeko-1 cells
(expressing
CD19) and to T cells expressing nfP2X7 CAR (data shown for anti-CD19 bridging
molecule only).
[0570] Table 4: Binding capacity of new CD19 BRiDGE variants to the CD19
positive cell line JeKo-1.
JeKo-1 cells were incubated with different 0019 BRiDGE variants at saturating
concentrations. After thorough washing, either a secondary anti-HIS-tag
antibody or a
single-domain antibody BILO3s (conjugated with AF647) staining at saturating
concentrations was used to semi-quantify the binding capacity to 0D19 on JeKo-
1. The
MFI intensity correlates with the number of secondary bound antibodies bound
to the
CD19 BRiDGE variants that are only detected if they are bound to the JeKo-1
cells.
MFI: median fluorescence intensity. BLItz: Bio-Layer interferometry (BLI)
technology
using HIS-tag identification tips. MFIR: median fluorescence intensity ratio
corresponds
to a relative index that is calculated by the testing condition MFI divided by
the control
MFI (HIS-tag or BIL03s) This corresponds to the MFI measured without the 0D19
BRiDGE primary staining (=isotype control) and the testing condition with the
CD19
BRiDGE and subsequent staining with the secondary antibody. The higher the
MFIR,
the stronger the binding. Targeting 0019 via a tafasitamab-based CD19 BRiDGE
shows the highest binding capacity to CD19 with the original E200 BRiDGE.
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MFI HisTag MF1131103 MFIR MFIR BIL03
MFIR BIL03 to
FITC AF647 HisTag FITC AF647 BLItz
HisTag
B1 3.21
3.19 4.168831169 6.934782609 0.1865 1.663483679
B2 3.47
4.17 4.506493506 9.065217391 0.1861 2.011590026
B3 3.63
5.43 4.714285714 11.80434783 0.2941 2.503952569
B4 3.89
9.02 5.051948052 19.60869565 0.1344 3.881412764
B5 3.4
5.5 4.415584416 11.95652174 0.2225 2.707800512
B6 3.33 5.29 4.324675325
11.5 0.1863 2.659159159
B7 3.31
7.57 4.298701299 16.45652174 0.2533 3.828254302
B8 3.58
10.6 4.649350649 23.04347826 0.1661 4.956278844
B9 3 4.82 3.896103896 10.47826087 0.2446
2.68942029
B10 3.27
7.71 4.246753247 16.76086957 0.2415 3.946749103
B11 3.08 6.43
4 13.97826087 0.2553 3.494565217
B12 3.36
9.08 4.363636364 19.73913043 0.2533 4.523550725
B13 2.99 5.28 3.883116883 11.47826087 0.2805
2.95594009
B14 3.12
7.27 4.051948052 15.80434783 0.2564 3.900431996
B15 2.91
8.58 3.779220779 18.65217391 0.1317 4.935454953
B16 2.86 8.08 3.714285714 17.56521739 0.2583
4.72909699
B17 2.98
9.3 3.87012987 20.2173913 0.1844 5.223956814
B18 3.03
10.9 3.935064935 23.69565217 0.3574 6.021667384
B19 2.7 4.9 3.506493506 10.65217391 0.2721
3.03784219
B20 2.61
4.17 3.38961039 9.065217391 0.1823 2.674412794
B21 2.56
4.06 3.324675325 8.826086957 0.2726 2.654721467
B22 2.54 4.85 3.298701299 10.54347826
0.23 3.196251284
B23 2.39
6.82 3.103896104 14.82608696 0.2123 4.776605421
B24 2.47
8.45 3.207792208 18.36956522 0.216 5.726544622
B25 2.32
5 3.012987013 10.86956522 0.2368 3.607571214
B26 2.36
5.53 3.064935065 12.02173913 0.232 3.922347089
B27 2.5
9.6 3.246753247 20.86956522 0.1628 6.427826087
B28 2.38
8.78 3.090909091 19.08695652 0.2021 6.175191816
B29 2.33
7.44 3.025974026 16.17391304 0.184 5.345027057
B30 2.35
7.48 3.051948052 16.26086957 0.2042 5.328029602
B31 2.13 5.83 2.766233766 12.67391304
0.25 4.581649316
B32 2.3
8.99 2.987012987 19.54347826 0.1712 6.542816635
B33 2.25
7.12 2.922077922 15.47826087 0.1655 5.297004831
B34 2.19
7.04 2.844155844 15.30434783 0.2197 5.380980743
B35 2.31 9.49
3 20.63043478 0.2333 6.876811594
B36 2.29
9.15 2.974025974 19.89130435 0.2135 6.68834251
CD19
PURIFIED 3.467532468 25.2173913
BRIDGE 2.67 11.6
7.2724312
NO BRIDGE 0.77 0.46 1 1
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Table 5: Binding capacity of the new CD19 BRiDGE variants to nfP2X7 CAR 1.
nfP2X7 CAR T cells were incubated with different BRiDGE variants at saturating

concentrations. After thorough washing, secondary anti-HIS-tag antibody
staining at
saturating concentrations was used to semi-quantify the binding capacity of
nfP2X7
CAR T cells to the E200 tag variant on the CD19 BRiDGE. The MFI intensity
correlates
with the number of secondary bound antibodies bound to the BRiDGEs that are
only
detected if they are bound to the CAR T cells. EGFR was used as a marker gene
to
detect the CAR expression. Thus, the EGFR+ T cell population was defined as
the CAR
expressing cells and the EGFR- T cell population was defined as the CAR
negative
subset. MFI: median fluorescence intensity. EGFR: epidermal growth factor
receptor.
BLItz: Bio-Layer interferometry (BLI) technology using HIS-tag identification
tips. HIS-
tag MFI Ratio: relative index that is calculated by the testing condition MFI
divided by
the control MFI, which corresponds to the MFI measured on EGFR+ T cells
(defined as
CAR positive cells) divided by the MFI measured on the EGFR- T cells (defined
as CAR
negative cells). Only CAR positive cells should be able to specifically bind
the BRiDGE
molecules via the E200 tag (nfP2X7-derived CAR targeted epitope). The higher
the
MFIR, the stronger the binding. Some of the new BRiDGE variants lead to a
significantly
improved binding to the nfP2X7 targeted CAR.
MFI HisTag FITC HisTag MFI HisTag MFI BLItz
MFI HisTag
in EGFR+ Ratio of Ratio
FITC in EGFR-
Population EGFR+/EGFR- (background
Population
0.44)
B1 2.1 0.52 4.0
4.8 0.1865
B2 2.07 0.51 4.1
4.7 0.1861
B3 2.05 0.5 4.1
4.7 0.2941
B4 2.05 0.5 4.1
4.7 0.1344
B5 2.08 0.5 4.2
4.7 0.2225
B6 1.99 0.49 4.1
4.5 0.1863
B7 2.22 0.55 4.0
5.0 0.2533
B8 2.53 0.58 4.4
5.8 0.1661
B9 2.32 0.56 4.1
5.3 0.2446
B10 2.26 0.53 4.3 5.1
0.2415
B11 2 0.5 4.0
4.5 0.2553
B12 2.12 0.51 4.2
4.8 0.2533
B13 2.13 0.52 4.1
4.8 0.2805
B14 2.41 0.54 4.5
5.5 0.2564
B15 2.61 0.54 4.8
5.9 0.1317
B16 2.34 0.51 4.6
5.3 0.2583
B17 2.25 0.53 4.2 5.1
0.1844
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B18 2.25 0.53 4.2 5.1
0.3574
B19 2.16 0.53 4.1 4.9
0.2721
B20 2.11 0.51 4.1 4.8
0.1823
B21 2.19 0.54 4.1 5.0
0.2726
B22 2.21 0.55 4.0 5.0
0.23
B23 2.34 0.53 4.4 5.3
0.2123
B24 2.13 0.54 3.9 4.8
0.216
B25 2.28 0.57 4.0 5.2
0.2368
B26 2.17 0.52 4.2 4.9
0.232
B27 2.35 0.57 4.1 5.3
0.1628
B28 2.24 0.53 4.2 5.1
0.2021
B29 2.25 0.54 4.2 5.1
0.184
B30 2.12 0.49 4.3 4.8
0.2042
B31 2.67 0.68 3.9 6.1
0.25
B32 2.61 0.66 4.0 5.9
0.1712
B33 2.39 0.61 3.9 5.4
0.1655
B34 2.41 0.56 4.3 5.5
0.2197
B35 2.51 0.61 4.1 5.7
0.2333
B36 2.41 0.58 4.2 5.5
0.2135
CD19 PURIFIED
BRIDGE 1.2 0.44 2.7 2.7
NO BRIDGE 0.62 0.35 1.8
[0571] Table 6: Binding capacity of new the CD33 BRiDGE variants to the C033
positive cell line MOLM-13.
MOLM-13 cells were incubated with different CD33 BRiDGE variants at saturating

concentrations. After thorough washing, secondary anti-HIS-tag antibody or
single-
domain antibody BILO3s (conjugated with AF647) staining at saturating
concentrations
was used to semi-quantify the binding capacity to 0D33 on MOLM-13. The MFI
intensity
correlates with the number of secondary antibodies bound to the CD33 BRiDGE
variants that are only detected if they are bound to the MOLM-13 cells. MFI:
median
fluorescence intensity. BLItz: Bio-Layer interferometry (BLI) technology using
HIS-tag
identification tips. MFIR: median fluorescence intensity ratio corresponds to
a relative
index that is calculated by the testing condition MFI divided by the control
MFI (HIS-tag
or BIL03s), which corresponds to the MFI measured without the CD33 BRiDGE
primary
staining (=isotype control) and the testing condition with CD33 BRiDGE and
subsequent
staining with the secondary antibody. The higher the MFIR, the stronger the
binding.
There is a significant increase in the BILO3s MFIR with the new BRiDGE
variants
compared to the old variants for the targeting of 0D33 with lintuzumab-based
BRiDGEs.
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MFI HisTag MFI Bil03 MFIR HisTag MFIR
BIL03 MFIR BIL03
FITC AF647 FITC AF647 BLItz to
HisTag
B1 6.11 25.6 9.119402985
40 0.1865 4.386252046
B2 5.59 23.8 8.343283582
37.1875 0.1861 4.457177996
B3 6.76 27.6 10.08955224
43.125 0.2941 4.274223373
B4 5.53 24.3 8.253731343
37.96875 0.1344 4.600192134
B5 5.86 24.8 8.746268657
38.75 0.2225 4.430460751
B6 6.25 25.8 9.328358209
40.3125 0.1863 4.3215
B7 5.52 24.7 8.23880597
38.59375 0.2533 4.684386322
B8 6.53 29.7 9.746268657
46.40625 0.1661 4.761437596
B9 6.33 28.7 9.447761194
44.84375 0.2446 4.746494866
B10 6.86 31.4 10.23880597
49.0625 0.2415 4.791818513
B11 7.39 34.2 11.02985075
53.4375 0.2553 4.844807172
B12 6.08 26.9 9.074626866
42.03125 0.2533 4.631733141
B13 6.78 28.5 10.11940299
44.53125 0.2805 4.400580752
B14 6.68 28 9.970149254
43.75 0.2564 4.388098802
B15 7.64 31.7 11.40298507
49.53125 0.1317 4.343709097
B16 6.27 28.7 9.358208955
44.84375 0.2583 4.791915869
B17 7.36 32.6 10.98507463
50.9375 0.1844 4.636973505
B18 5.79 24.2 8.641791045
37.8125 0.3574 4.375539724
B19 5.35 21.5 7.985074627
33.59375 0.2721 4.207067757
B20 5.67 21.5 8.462686567
33.59375 0.1823 3.969631834
B21 6.04 25.8 9.014925373
40.3125 0.2726 4.471750828
B22 4.61 19.8 6.880597015
30.9375 0.23 4.496339479
B23 6.82 28.3 10.17910448
44.21875 0.2123 4.344070748
B24 6.25 26.7 9.328358209 41.71875 0.216
4.47225
B25 7.29 28.5 10.88059701
44.53125 0.2368 4.092721193
B26 5.75 25.9 8.582089552 40.46875 0.232 4.71548913
B27 5.55 25.1 8.28358209
39.21875 0.1628 4.734515766
B28 6.33 27.8 9.447761194
43.4375 0.2021 4.597650079
B29 5.38 23.4 8.029850746 36.5625 0.184 4.553322491
B30 6.29 27.9 9.388059701
43.59375 0.2042 4.643531399
B31 7.09 30.3 10.58208955 47.34375 0.25 4.473950987
B32 6.69 27.7 9.985074627
43.28125 0.1712 4.334594544
B33 6.96 29.8 10.3880597
46.5625 0.1655 4.482309626
B34 6.98 29.8 10.41791045
46.5625 0.2197 4.469466332
B35 6.62 27.4 9.880597015
42.8125 0.2333 4.33298716
B36 6.72 29 10.02985075
45.3125 0.2135 4.517764137
CD33
PURIFIED 10.62686567 18.125
BRIDGE 7.12 11.6
1.705582865
NO BRIDGE 0.67 0.64 1 1
[0572] Table 7: Binding capacity of new CD33 BRiDGE variants to nfP2X7 CAR
1.
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nfP2X7 CAR T cells were incubated with different BRiDGE variants at saturating

concentrations. After thorough washing, secondary anti-HIS-tag antibody
staining at
saturating concentrations was used to semi-quantify the binding capacity of
nfP2X7
CAR T cells to the E200 tag variant on the CD33 BRiDGE. The MFI intensity
correlates
with the number of secondary bound antibodies bound to the BRiDGEs that are
only
detected if they are bound to the CAR T cells. EGFR was used as a marker gene
to
detect the CAR expression. Thus, the EGFR+ T cell population was defined as
the CAR
expressing cells and the EGFR- T cell population was defined as the CAR
negative
subset. MFI: median fluorescence intensity. EGFR: epidermal growth factor
receptor.
BLItz: Bio-Layer interferometry (BLI) technology using HIS-tag identification
tips. HIS-
tag MFI Ratio: relative index that is calculated by the testing condition MFI
divided by
the control MFI, which corresponds to the MFI measured on EGFR+ T cells
(defined as
CAR positive cells) divided by the MFI measured on the EGFR- T cells (defined
as CAR
negative cells). Only CAR positive cells should be able to specifically bind
the BRiDGE
molecules via the E200 tag (nfP2X7-derived CAR targeted epitope). The higher
the
MFIR, the stronger the binding. Some of the new BRiDGE variants lead to a
significantly
improved binding to the nfP2X7 targeted CAR.
MFI HisTag MFI HisTag HisTag MFI HisTag MFI
BLItz
FITC in EGFR+ FITC in EGFR- Ratio of Ratio
Population Population
EGFR+/EGFR- (background
0.4)
B1 1.08 0.44 2.5 2.7
0.1865
B2 1.11 0.45 2.5 2.8
0.1861
B3 1.04 0.44 2.4 2.6
0.2941
B4 1.68 0.54 3.1 4.2
0.1344
B5 1.4 0.5 2.8 3.5
0.2225
B6 1.96 0.57 3.4 4.9
0.1863
B7 1.45 0.49 3.0 3.6
0.2533
B8 1.34 0.47 2.9 3.4
0.1661
B9 1.16 0.42 2.8 2.9
0.2446
B10 1.99 0.57 3.5 5.0
0.2415
B11 1.2 0.45 2.7 3.0
0.2553
B12 1.55 0.5 3.1 3.9
0.2533
B13 1.35 0.47 2.9 3.4
0.2805
B14 1.44 0.49 2.9 3.6
0.2564
B15 1.5 0.49 3.1 3.8
0.1317
B16 1.83 0.54 3.4 4.6
0.2583
B17 1.58 0.5 3.2 4.0
0.1844
B18 1.79 0.52 3.4 4.5
0.3574
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B19 1.29 0.45 2.9 3.2
0.2721
B20 1.29 0.45 2.9 3.2
0.1823
B21 1.14 0.41 2.8 2.9
0.2726
B22 1.44 0.47 3.1 3.6
0.23
B23 1.71 0.51 3.4 4.3
0.2123
B24 1.82 0.52 3.5 4.6
0.216
B25 1.82 0.54 3.4 4.6
0.2368
B26 1.22 0.43 2.8 3.1
0.232
B27 1.2 0.43 2.8 3.0
0.1628
B28 1.58 0.49 3.2 4.0
0.2021
B29 1.73 0.51 3.4 4.3
0.184
B30 1.63 0.49 3.3 4.1
0.2042
B31 1.34 0.45 3.0 3.4
0.25
B32 1.59 0.48 3.3 4.0
0.1712
B33 1.69 0.5 3.4 4.2
0.1655
B34 1.31 0.43 3.0 3.3
0.2197
B35 2.15 0.56 3.8 5.4
0.2333
B36 1.65 0.5 3.3 4.1
0.2135
CD33 PURIFIED
BRIDGE 1.58 0.4 40 4.0
NO BRIDGE 0.23 0.4 0.6
[0573] Subsequently, the bridging molecules were assessed functionally, for
their
ability to induce cell death (cytotoxicity). Experiments conducted were
similar to those
described in Examples 11-15.
[0574] All bridging molecules tested demonstrated an ability to induce cell
killing.
Thus, the data indicate that the bridging molecule successfully re-directed
anti-nfP2X7
CAR T cells to either 0019 or CD33 antigen expressed by JeKo-1 cells or MOLM-
13
cells, respectively. Representative data are shown in Figure 36 (A: showing
cell killing of
JeKo-1 cells by anti-CD19 bridging molecule + anti-nfP2X7 CAR T cells; B:
showing cell
killing of MOLM-13 cells by anti-0D33 bridging molecule + anti-nfP2X7 CAR T
cells).
Example 17 - in vivo efficacy of two component therapeutic system of the
invention
[0575] 6-8-week-old NSG mice were inoculated with the single cell sorted
reporter
cell line JeKo-1_LUC_eGFP at 1x106 cells per mouse via tail vein injection on
day 0. On
day 7, mice underwent bioluminescence imaging (BLI) to determine JeKo-1
engraftment. Further, BLI was used to quantify the leukaemic load and
treatment was
initiated on day 7.
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[0576] Mice were divided into the following treatment groups:
1. Tumour only (PBS)
2. Activated untransduced T cells
3. Bridging molecule (anti-CD19 Fab based on tafasitamab with E200 epitope)
4. Activated untransduced T cells + bridging molecule
5. T cells expressing 3rd generation anti-CD19-CAR (antigen binding domain
derived from FMC63 and having general CAR architecture of: CD8-CD28-41BB-
CD3)
6. T cells expressing anti-nfP2X7 receptor CAR 1 (antigen binding domain 2-2-
1)
7. T cells expressing CAR1 + bridging molecule
8. T cells expressing anti-nfP2X7 receptor CAR2 (anti-nfP2X7 receptor CAR
antigen
binding domain 2-2-1)
9. T cells expressing CAR2 + bridging molecule
10. PBS control (duplicate)
[0577] On day 7, intraperitoneal administration of bridging molecule was
commenced
at a dose regimen of 3 x per week at 50 pg per mouse in the indicated groups.
Leukaemic burden was evaluated via BLI at the indicated time points and mouse
blood
was analysed on day 42 to detect cancer cells and immune cells in the
circulating
peripheral blood. The experimental design is illustrated in Figure 37.
[0578] The bridging molecule used comprised similar architecture to the
molecules
described in Example 14 (ie, having a targeting moiety comprised of a Fab
derived from
tafasitamab), and a tumour-specific antigen epitope moiety comprising an E200
peptide
based on the SEQ ID NO: 143 and 145.
[0579] The positive control (group 5) comprised T cells expressing a 3rd
generation
anti-CD19 CAR. The CAR comprised an antigen binding domain derived from FMC63
as described elsewhere herein (eg SEQ ID NO: 31 without the 6xHIStag and 32 in
light-
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heavy orientation) and having the domains CD8-CD8TM-CD28-41BB-CD3zeta, a
commonly used conventional CAR domain structure.
[0580] The ability of the bridging molecule to redirect T cells expressing two
different
nfP2X7 receptor CARs was assessed. Briefly nfP2X7 receptor CAR1 and nfP2X7
receptor CAR2 comprised the general structure of a CAR, as described above in
example 8 (ie having an antigen binding domain comprising the 2-2-1 sdAb,
capable of
binding an E200 epitope).
[0581] Figure 38 shows bioluminescence (as a marker of tumour burden) for each
of
the treatment groups. The results indicate that mice administered T cells
expressing
anti-nfP2X7 receptor CAR + anti-CD19 bridging molecule, had similarly low
levels of
tumour burden as compared to mice that received T cells expressing an anti-
CD19
CAR.
[0582] These results demonstrate that in an in vivo setting, the bridging
molecules of
the present invention can be used to redirect a CAR T cell to bind to an
alternative
target. In this case, CAR T cells directed to nfP2X7 receptor were
successfully
redirected to the CD19 antigen on cancer cells and achieved similar levels of
therapeutic efficacy as CAR T cells with an antigen binding domain for binding
CD19.
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